Compositions and methods for inhibition of nucleic acids function

ABSTRACT

The invention relates generally to compositions and methods for inhibiting the function of target nucleic acids by sequence specific binding. The compositions and methods can be used for inhibition of micro RNAs and other relatively short non-coding RNAs.

This application claims the benefit of U.S. Provisional Application Ser.Nos. 61/323,664 filed Apr. 13, 2010 and 61/430,005 filed Jan. 5, 2011.The provisional applications are incorporated by reference herein intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 4, 2011, isnamed LT0183US.txt and is 77,601 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods forinhibiting the function of target nucleic acids by sequence specificbinding. The compositions and methods can be used for inhibition ofmicro RNAs and other short non-coding RNAs.

BACKGROUND INFORMATION

Recent evidence suggests that the human transcriptome is not onlysignificantly larger than previously recognized but also it is primarilycomposed of functional RNA transcripts which are not translated intoproteins known as non-coding RNAs (ENCODE Project Consortium, Nature2007, v.447, p. 799-816). Among the more intensely studied class ofendogenously expressed non-coding RNAs are the micro-RNAs (miRNAs).Mature miRNAs are relatively small (21-23 nucleotides) RNA duplexes thatact as translational repressors of protein expression. The guide strandof a miRNA unites with Argonaute family proteins (Ago) to formRNA-Induced Silencing Complexes (RISC) in the cell. Thesesequence-specific ribonucleoprotein complexes bind target mRNAstypically in the 3′UTR and can subsequently silence gene expressioneither through directed mRNA degradation or by simply sequestering thetarget mRNA in an ineffectual form (Lee et al., Cell 1993, v.75, p.843-854; Bartel, Cell 2009 v. 136, p. 215-233). It has been demonstratedthat miRNA based regulation plays a significant role in routine cellularprocesses including metabolism (Esau et al, Cell Met. 2006, v.3, p87-98), development (Carthew et al., Cell 2009, v.137, p. 273-282), andeven apoptosis (Cheng et al, Nucl. Acids Res. 2005, v.33, p1290-1297).Further research has revealed that miRNAs play critical roles in diversedisease processes such as hepatitis C (Jopling et al., Science 2005,v.309, p. 1577-1581), diabetes (Poy et al., Nature 2004, v.432, p.226-230), and most notably multiple cancer types (Hammond, Can. Chemo.Pharma. 2006 v.58, s63-s68; Calin et al., Cancer Res. 2006, v.66, p.7390-7394) including leukemia (Calin et al., PNAS 2002, v.101, p.2999-3004) and glioma (Corsten et al., Cancer Res. 2007, v.67, p.8994-9000). In addition, miRNA discovery has far surpassed miRNAphenotypic identification thus creating a “validation gap” for miRNAfunction (Griffiths-Jones et al., Nucl. Acids Res. 2006, v.34,p.D141-D144). Over one thousand miRNAs have now been identified inanimals, but only a few individual miRNAs have been linked to specificfunctions.

Given the range and degree of effects that miRNAs have on cellularprocesses and that a single miRNA can modulate multiple gene products(Selbach et al., Nature 2008, v.455, p. 58-63), miRNAs have becomeattractive targets both for loss of function studies in vitro (to studymiRNA function and mechanism) and potential therapeutic applications invivo. To this end, research groups both public and private have soughtto develop highly potent miRNA inhibitors (antisense oligos which bindto complementary miRNAs and selectively block silencing in cellextracts, in cultured cells, and in vivo) by utilizing one of threebasic approaches: (i) antisense (AS)-based oligonucleotides that employchemically modified sugars, bases, phosphate backbones, and/or terminalconjugates (Krutzfeldt et al., Nucl. Acids Res. 2007 v.35, p. 2885-2892,Horwich et al., Nat. Protocols, 2008 v.3, p. 1537-1549, Davis et al.,Nucl. Acids Res. 2006, v.34, p. 2294-2304); (ii) long (>34 nucleotides)oligonucleotides wherein the reverse complement (AS) strand to the miRNAis flanked on both the 3′ and 5′ end with 12-16 nucleotides which areintended to form hairpin loops (Vermeulen et al., RNA 2007, v.13, p.723-730); and (iii) Tandemic antisense RNA produced from DNA vectorswith multiple miRNA binding sites per unit which behave as decoy targetsfor endogenous miRNAs (“miRNA sponges”, Ebert et al, Nature Methods2007, v.4, p. 721-'726). Further, 2′ O-Me oligonucleotides have beenproduced to be resistant to cleavage by RISC and other cellularribonucleases (Hutvagner, et al., PloS Biol. 2004, v. 2, p. E98;Meister, et al., RNA 2004, v.10, p. 544-550). Oligonucleotides have alsobeen made that combine 2′-deoxy and locked nucleic acid (LNA)nucleotides (Lecellier et al., Science 2005, v. 308, p. 557-560).Oligonucleotides have been made that contain all 2′-O-methoxyethylnucleotides and oligonucleotides incorporating pyrimidines bearing2′-O-fluoromodifications (Essau et al., Cell Metab. 2006, v. 3, p.87-89; Davis et al, Nucleic Acids Res. 2006, v. 34, p. 2294-2304).Further, nuclease resistant phosphorothioate backbone linkages incombination with ribose modifications have also been employed incultured cells and in vivo in mice and non-human primates (Essau et al.,Cell Metab. 2006, v. 3, p. 87-89; Elmén et al., Nature, 2008, v. 452, p.896-899).

Life Technologies currently offers miRNA inhibitors with proprietarymodifications (AntiMiRs), Exiqon offers LNA-modified short antisenseinhibitors, and Dharmacon/ThermoFisher offers 2′-OMe antisenseinhibitors with hairpin structure motifs at both the 3′ and 5′ ends (>55nt). The invention discussed herein offers several novel and uniquedesigns for miRNA inhibitors that enable superior miRNA inhibition.

SUMMARY OF THE INVENTION

This disclosure provides compositions and methods for inactivatingnucleic acid molecules. Methods of the invention involve, in part, theuse of molecules with nucleic acid regions with sequence complementarityto the nucleic acid molecules which is the subject of desiredinactivation (i.e., a target nucleic acid molecule). Methods of theinvention can be used for inactivation of relatively short regulatorynon-coding RNAs, such as micro RNAs (miRNAs), Piwi-interacting RNAs(piRNAs), snoRNAs, snRNAs, moRNAs, PARs, sdRNAs, tel-sRNAs, crasiRNAs,and small interfering RNAs (siRNAs). Methods of the invention can alsobe used for long non-coding RNAs (long ncRNAs), traditional non-codingtRNAs and ribosomal RNA (rRNA).

In general methods of the invention include those which comprise one ormore of the following steps: contacting the target RNA or DNA moleculewith one or more of the composite nucleic acid inhibitory molecules setout below:

Some embodiments of the invention are directed to composite nucleic acidinhibitory molecules which comprise one or more of the followingregions: (a) a first homologous region, (b) a loop, (c) a secondhomologous region, and (d) a target binding nucleic acid segment whichis greater than 6 nucleotides in length, wherein the loop connects thefirst homologous region and the second homologous region and is ofsufficient length to allow for the first homologous region and thesecond homologous region to hybridize to one another to form adouble-stranded structure, and wherein the first homologous region andthe second homologous region share sufficient sequence homology to allowfor hybridization under physiological conditions. In some embodiments,the target is a long non-coding RNA or a short non-coding RNA. In someembodiments, the short non-coding RNA is chosen from a microRNA (miRNA),a Piwi-interacting RNA (piRNA), and a small interfering RNA (siRNA). Insome embodiments, the loop is a non-nucleotide loop. In someembodiments, the target binding nucleic acid segment is about 60 toabout 100% complementary to all or a portion of at least one targetnucleic acid. In some embodiments, the first homologous region or thesecond homologous region is between from about 3 nucleotides to about 30nucleotides in length. In some embodiments, the first homologous regionis between about 4 and about 10 nucleotides in length. In someembodiments, the backbone of the loop comprises covalently bonded carbonatoms. In some embodiments, the backbone of the loop comprisescovalently bonded carbon and oxygen atoms. In some embodiments, the loopis chosen from polyethylene glycol, C2-C18 alkane diol, styrene,stilbene, triazole, tetrazole, nucleic acid, poly abasic nucleoside,polysaccharide, peptide, polyamide, hydrazone, oxyimine, polyester,disulfide, polyamine, polyether, peptide nucleic acid, cycloalkane,polyalkene, aryl, derivatives thereof and any combination thereof. Insome embodiments, the homologous region is separated from the targetbinding nucleic acid segment by at least one nucleotide having a purinebase, a pyrimidine base, or no base (abasic). In some embodiments, thenucleotides that comprise the target-binding nucleic acid segment aremodified and those modifications are chosen from, DNA, RNA, 2′OMe,2′OAllyl, 2′O-propargyl, 2′O-alkyl, 2′ fluoro, 2′ arabino, 2′ xylo, 2′fluoro arabino, phosphorothioate, phosphorodithioate, phosphoroamidates,2′Amino, 5-alkyl-substituted pyrimidine, 5-halo-substituted pyrimidine,alkyl-substituted purine, halo-substituted purine, bicyclic nucleotides,2′MOE, LNA, LNA-like molecules and derivatives thereof. In someembodiments, the polyethylene glycol is a polyethylene glycol derivativeand wherein the polyethylene glycol derivative is hexa-ethylene glycolor penta-ethylene glycol. In some embodiments, the loop is C12 alkanediol. In some embodiments, the molecule is modified and the modificationadds at least one amine, imine, guanidine, or aromatic aminoheterocycle. In some embodiments, the nucleotides in the homologousregion and/or spacer comprises at least one 2′ O-alkyl, LNA, 2′ fluoro,arabino, 2′ xylo, 2′ fluoro arabino, phosphorothioate,phosphorodithioate, 2′ amino, bicyclic nucleotide, 5-alkyl-substitutedpyrimidine, 5-halo-substituted pyrimidine, alkyl-substituted purine, orhalo-substituted purine, halo-substituted purine, bicyclic nucleotides,2′MOE, LNA, and derivatives thereof. In some embodiments, the targetbinding nucleic acid segment is between from about 10 nucleotides toabout 100 nucleotides in length. In some embodiments, the target bindingnucleic acid segment is between from about 15 to about 25 nucleotides inlength. In some embodiments, the first homologous region, the secondhomologous region and the loop form a stem loop structure and the stemloop structure is on the 5′ end of the nucleic acid inhibitory molecule.In some embodiments, the first homologous region, the second homologousregion and the loop form a stem loop structure and the stem loopstructure is on the 3′ end of the nucleic acid inhibitory molecule. Insome embodiments, the composite nucleic acid inhibitory molecule alsoincludes a second stem loop structure at the 5′ end of the nucleic acidinhibitory molecule. In some embodiments, the entire molecule is betweenabout 20 and about 150 nucleotides in length. In some embodiments, theinhibitory molecule is modified and the modification is a covalentlylinked conjugate that enhances cell penetration, endocytosis,facilitated diffusion, tissue localization, inhibitor detection, orcellular trafficking of the modified nucleic acid inhibitory molecule.In some embodiments, the inhibitory molecule further comprises one ormore nucleotides on either end of the molecule.

Some embodiments of the invention are directed to multimeric nucleicacid inhibitory molecules which comprises at least one or more of thefollowing regions: (a) a first oligonucleotide and (b) a secondoligonucleotide, wherein the first oligonucleotide comprises a targetnucleic acid molecule binding region, and two regions which will nothybridize to the target nucleic acid molecule, wherein the secondoligonucleotide comprises two regions which will hybridize underphysiological conditions to at least two of the two regions of the firstoligonucleotides which will not hybridize to the target nucleic acidmolecule, and wherein the multimeric nucleic acid inhibitory moleculecomprises both single-stranded and double-stranded regions. In someembodiments, the sequence that will not hybridize to the target nucleicacid molecule is separated from the target binding nucleic acid segmentby at least one nucleotide having a purine base, a pyrimidine base, orno base (abasic). In some embodiments, the target nucleic acid moleculeis a long non-coding RNA or a short non-coding RNA. In some embodiments,the first oligonucleotide is between from about 30 nucleotides to about200 nucleotides in length. In some embodiments, the secondoligonucleotide is between from about 6 nucleotides to about 50nucleotides in length. In some embodiments, the single-stranded regionis capable of binding a target nucleic acid molecule under physiologicalconditions.

In some embodiments, the single-stranded region is complementary to allor a portion of the target nucleic acid. In some embodiments, the shortnon-coding RNA is chosen from a microRNA (miRNA), Piwi-interacting RNA,and small interfering RNA, a messenger RNA, and a ribosomal RNA. In someembodiments, the nucleic acid molecule comprises at least onemodification chosen from, DNA, RNA, 2′OMe, 2′OAllyl, 2′O-propargyl,2′O-alkyl, 2′ fluoro, 2′ arabino, 2′ xylo, 2′ fluoro arabino,phosphorothioate, phosphorodithioate, 2′ amino, 5-alkyl-substitutedpyrimidine, 5-halo-substituted pyrimidine, alkyl-substituted purine,halo-substituted purine, bicyclic nucleotides, 2′MOE, LNA, andderivatives thereof. In some embodiments, the molecule further comprisesone or more nucleotides on either end of the molecule. In someembodiments, the target binding nucleic acid segment is about 60 toabout 100% complementary to all or a portion of at least one targetnucleic acid.

A further aspect of the invention is a method for inhibiting thefunction of a target RNA or DNA molecule, the method comprisingcontacting the target RNA or DNA molecule with at least one compositenucleic acid inhibitory molecule which comprises the following regions:a first homologous region, a loop, a second homologous region, and atarget binding nucleic acid segment which is greater than 6 nucleotidesin length, wherein the loop connects the first homologous region and thesecond homologous region and is of sufficient length to allow for thefirst homologous region and the second homologous region to hybridize toone another to form a double-stranded structure, and wherein the firsthomologous region and the second homologous region share sufficientsequence homology to allow for hybridization under physiologicalconditions.

A further aspect of the invention is a method for inhibiting thefunction of a target RNA or DNA molecule, the method comprisingcontacting the target RNA or DNA molecule with at least one multimericnucleic acid inhibitory molecule having at least a first oligonucleotideand a second oligonucleotide. In many instances, the firstoligonucleotide comprises a target nucleic acid molecule binding regionand two regions which will not hybridize to the target nucleic acidmolecule, wherein the second oligonucleotide comprises two regions whichwill hybridize under physiological conditions to at least two of the tworegions of the first oligonucleotides which will not hybridize to thetarget nucleic acid molecule, and wherein the multimeric nucleic acidinhibitory molecule comprises both single-stranded and double-strandedregions.

A further aspect of the invention is a method for producing a multimericnucleic acid inhibitory molecule, including obtaining at least a firstoligonucleotide and a second oligonucleotide, wherein the firstoligonucleotide comprises a target nucleic acid molecule binding region,and two or more regions which will not hybridize to the target nucleicacid molecule, wherein the second oligonucleotide comprises two regionswhich will hybridize under physiological conditions to at least two ofthe two or more regions of the first oligonucleotide which will nothybridize to the target nucleic acid molecule; admixing equal amounts ofthe first and second oligonucleotides in an appropriate buffer andallowing annealing of the two molecules, wherein the resultingmultimeric nucleic acid inhibitory molecule comprises bothsingle-stranded and double-stranded regions.

A further aspect of the invention is a composite nucleic acid inhibitorymolecule which includes a 5′ and/or 3′ non-nucleotide loop, and a targetbinding nucleic acid segment which is greater than 6 nucleotides inlength, wherein the target binding nucleic acid segment is modified toincrease binding affinity to its target. In some embodiments, thenon-nucleotide loop is chosen from polyethylene glycol (PEG3-PEG10),C2-C18 alkane diol, styrene, stilbene, triazole, tetrazole, nucleicacid, poly abasic nucleoside, polysaccharide, peptide, polyamide,hydrazone, oxyimine, polyester, disulfide, polyamine, polyether, peptidenucleic acid, cycloalkane, polyalkene, aryl, any combination thereof,and derivatives thereof. In some embodiments, the loop is separated fromthe target binding nucleic acid segment by at least one nucleotidehaving a purine base, a pyrimidine base, or no base (abasic). In someembodiments, the target nucleic acid molecule is a long non-coding RNAor a short non-coding RNA. In some embodiments, the short non-coding RNAis chosen from a microRNA (miRNA), a Piwi-interacting RNA (piRNA), and asmall interfering RNA (siRNA), snoRNAs, snRNAs, moRNAs, PARs, sdRNAs,tel-sRNAs, crasiRNAs, and small interfering RNAs (siRNAs). Methods ofthe invention can also be used for long non-coding RNAs (long ncRNAs),traditional non-coding tRNAs and ribosomal RNA (rRNA).

In some embodiments, the nucleotides in the target binding nucleic acidsegment comprises at least one 2′ O-alkyl, LNA, 2′ fluoro, 2′ arabino,2′ xylo, 2′ fluoro arabino, phosphorothioate, phosphorodithioate, 2′amino, bicyclic nucleotide, 5-alkyl-substituted pyrimidine,5-halo-substituted pyrimidine, alkyl-substituted purine, orhalo-substituted purine, halo-substituted purine, bicyclic nucleotides,2′MOE, LNA, and derivatives thereof. In some embodiments, the targetbinding nucleic acid segment is about 60 to about 100% complementary toall or a portion of at least one target nucleic acid.

A further aspect of the invention is a method for inhibiting thefunction of a target RNA or DNA molecule, the method comprisingcontacting the target RNA or DNA molecule with at least one compositenucleic acid inhibitory molecule which includes a 5′ and/or 3′non-nucleotide loop, and a target binding nucleic acid segment which isgreater than 6 nucleotides in length, wherein the target binding nucleicacid segment is modified to increase binding affinity to its target.

A further aspect of the invention is a method for treatment of adisease, comprising administering at least one composite nucleic acidinhibitory molecule which includes a 5′ and/or 3′ non-nucleotide loop,and a target binding nucleic acid segment which is greater than 6nucleotides in length, wherein the target binding nucleic acid segmentis modified to increase binding affinity to its target, in an amounteffective to treat a disease, wherein treatment of the disease comprisesreducing the symptoms of a disease. In some embodiments, the method alsoincludes complexing the inhibitory molecule with a cellular deliveryagent. In some embodiments, the disease is chosen from cancer,Alzheimer's disease, diabetes, and viral infections.

A further aspect of the invention is a method for treatment of adisease, comprising administering at least one composite nucleic acidinhibitory molecule which comprises the following regions: a firsthomologous region, a loop, a second homologous region, and a targetbinding nucleic acid segment which is greater than 6 nucleotides inlength, wherein the loop connects the first homologous region and thesecond homologous region and is of sufficient length to allow for thefirst homologous region and the second homologous region to hybridize toone another to form a double-stranded structure, and wherein the firsthomologous region and the second homologous region share sufficientsequence homology to allow for hybridization under physiologicalconditions, in an amount effective to treat a disease, wherein treatmentof the disease comprises reducing the symptoms of a disease. In someembodiments, the method also includes complexing the inhibitory moleculewith a cellular delivery agent. In some embodiments, the disease ischosen from cancer, Alzheimer's disease, diabetes, and viral infections.

A further aspect of the invention is a method for treatment of adisease, comprising administering at least one a multimeric nucleic acidinhibitory molecule having at least a first oligonucleotide and a secondoligonucleotide, wherein the first oligonucleotide comprises a targetnucleic acid molecule binding region, and two regions which will nothybridize to the target nucleic acid molecule, wherein the secondoligonucleotide comprises two regions which will hybridize underphysiological conditions to at least two of the two regions of the firstoligonucleotides which will not hybridize to the target nucleic acidmolecule, and wherein the multimeric nucleic acid inhibitory moleculecomprises both single-stranded and double-stranded regions, in an amounteffective to treat a disease, wherein treatment of the disease comprisesreducing the symptoms of a disease. In some embodiments, the method alsoincludes complexing the molecule with a cellular delivery agent. In someembodiments, the disease is chosen from cancer, Alzheimer's disease,diabetes, and viral infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematic structures of the hairpin (HP)-based miRNAinhibitors (anti-miR5) used in Examples 1-6. Two types of loops wereused: one all nucleotide and the other non-nucleotide (e.g., PEG3). Thetop part of the figure shows the nucleic acid molecules beforehybridization. The bottom part of the figure shows the structure whenbound to the target nucleic acid (e.g., a miRNA). As shown in thefigure, type 1 has a 5′ stem loop, type 2 has both a 5′ and 3′ stemloop, and Type 3 has a 3′ stem loop. All experiments were performed intriplicate.

FIG. 2 shows a schematic depiction of one of the assays used forevaluation of performance of the miRNA inhibitors (anti-miRs). Thefigure shows the reporter (firefly luciferase) expression in thepresence of endogenous microRNAs and exogenous anti-miRs. Anti-miRs bindand inhibit the miRNAs and thus increase the firefly luciferaseexpression. All experiments were performed in triplicate.

FIG. 3A and FIG. 3B show data derived from the Example 1 experiment inwhich the potency of the hairpin inhibitors HP#01-HP#09 was evaluatedusing the pMIR-REPORT™ miRNA expression reporter (miR21 or let7c targetcloned), along with two controls: a 22 nt miRNA inhibitor with complete2′-OMe modification (2′-OMe), and miRNA inhibitor X (Exiqon). FIG. 3A:miR21 target; FIG. 3B: let7c target. The expression induced by miRNAinhibition is the Average fold change and is calculated in the followingway: (fLuc activity anti-miR/βGal activity anti-miR)/(fLuc activitynegative/βGal activity negative). Note: the higher the bars—the strongerthe miRNA inhibition. All experiments were performed in triplicate.

FIG. 4A and FIG. 4B show data derived from the Example 2 experiment inwhich the efficiency of the hairpin inhibitors HP#05, HP#08, andHP#10-HP#31 was evaluated using pMIR-REPORT™ miRNA expression reporter(miR21 or let7c target cloned), along with three controls: a 22 nt miRNAinhibitor with complete 2′-OMe modification (2′-OMe), miRNA inhibitor X(Exiqon) and miRNA inhibitor Y (Dharmacon). FIG. 4A: miR21 target.Concentrations of miRNA inhibitors upon transfection: 3 nM (solid bar)and 0.3 nM (open bar). FIG. 4B: let7c target. Concentration of miRNAinhibitors upon transfection: 0.3 nM. Average fold change is calculatedin the following way: (fLuc activity anti-miR/βGal activityanti-miR)/(fLuc activity negative/βGal activity negative). Allexperiments were performed in triplicate.

FIG. 5 shows data derived from the Example 3 experiment in which thepotency of the hairpin inhibitors HP#05, HP#08, and HP#10-HP#31targeting let-7c miRNA was evaluated by quantification of the levels ofthe endogenous HMGA2 mRNA transcript expression using TAQMAN® geneexpression assays, along with three controls: a 22 nt miRNA inhibitorwith complete 2′-OMe modification (2′-OMe), miRNA inhibitor X (Exiqon)and miRNA inhibitor Y (Dharmacon). The concentration of miRNA inhibitorsupon transfection: 0.3, 3 and 30 nM. Increase of the HMGA2 mRNA levelsis shown relative to negative control-transfected samples. Allexperiments were performed in triplicate.

FIG. 6A and FIG. 6B show data derived from the Example 4 experiment inwhich the potency of the hairpin inhibitor HP#24 was evaluated byquantification of the fraction of the free endogenous miRNA (miR21 orlet7c) using a TAQMAN® assay. Three inhibitor controls were used: a 22nt miRNA inhibitor with complete 2′-OMe modification (2′-OMe), miRNAinhibitor X (Exiqon) and miRNA inhibitor Y (Dharmacon). FIG. 6A: let-7ctarget. FIG. 6B: miR-21 target. Concentration of miRNA inhibitors upontransfection: 3 and 30 nM. Decrease of the free miRNA levels (availablefor primer hybridization and thus detection) is shown relative tonegative control-transfected samples. All experiments were performed intriplicate.

FIG. 7A-FIG. 7C show data derived from the Example 5 experiment in whichthe efficiency of the hairpin inhibitors HP#79-HP#83 was evaluated usinga pMIR-REPORT™ miRNA expression reporter (miR21, let7a or let7c targetcloned), along with three controls: a 22 nt miRNA inhibitor withcomplete 2′-OMe modification (2′-OMe), miRNA inhibitor X (Exiqon) andmiRNA inhibitor Y (Dharmacon). FIG. 7A: miR21 target; FIG. 7B: let7atarget; FIG. 7C: let7c target. Concentrations of miRNA inhibitors upontransfection: 30, 3 and 0.3 nM. Average fold change is calculated in thefollowing way: (fLuc activity anti-miR/βGal activity anti-miR)/(fLucactivity negative/βGal activity negative). Note: the higher the bars—thestronger the miRNA inhibition. All experiments were performed intriplicate.

FIG. 8A-FIG. 8C show data derived from the Example 6 experiment in whichthe efficiency of the modified antisense oligonucleotide-basedinhibitors BTM#03-BTM#07, and BTM#22 was evaluated using a pMIR-REPORT™miRNA expression reporter (miR21, let7a or let7c target cloned), alongwith two controls: a 22 nt miRNA inhibitor with complete 2′-OMemodification (2′-OMe) and with miRNA inhibitor X (Exiqon). FIG. 8A:miR21 target; FIG. 8B: let7a target; FIG. 8C: let7c target.Concentration of miRNA inhibitors upon transfection: 3 nM. Average foldchange is calculated in the following way: (fLuc activity anti-miR/βGalactivity anti-miR)/(fLuc activity negative/βGal activity negative).Note: the higher the bars—the stronger the miRNA inhibition. Allexperiments were performed in triplicate.

FIG. 9 is a schematic showing formats of embodiments andinter-relationships of the inhibitors herein including hairpininhibitors (B1 and C1) and multimeric inhibitors (B2A, B2B, and C2).Regions of the inhibitors are designated as follows: NBR=non-bindingregion, S=Spacer, BR=binding region, HR=homology region, Loop=nucleotideor non-nucleotide loop. All experiments were performed in triplicate.

FIG. 10 shows the formation of inhibitors used in the Example 7experiment in which the formation of multimeric inhibitor complexes wasevaluated. The long 2′-OMe modified anti-miRs (elongated at 3′ and 5′ends-11 nt at the 5′ end and 12 nt at the 3′ end) and “bridging”oligonucleotide (21 nt) were combined and heated in the annealingbuffer, followed by slow cooling to RT. The top sequence is the 2′OMemodified long anti-mir21 (SEQ ID NO:139), the middle sequence is the“bridging” oligonucleotide (SEQ ID NO:140), the bottom sequence is themultimeric strand containing the target binding sequences (SEQ IDNO:141), and the “bridging” oligonucleotide (SEQ ID NO:140). Allexperiments were performed in triplicate.

FIG. 11 shows data derived from the Example 7 experiment assaying theinhibitory activity of antisense oligonucleotide-based multimericanti-miRs. #1, #2, and #3 refer to different annealing buffers. Buffer#1: 10 mM Tris-HCl pH 7.4, 100 mM NaCl. Buffer #2: 6 mM HEPES pH 7.4, 20mM potassium acetate, 0.4 mM Magnesium acetate. Buffer#3: 10 mM Tris-HClpH 8.0, 10 mM NaCl, 1 mM EDTA. 1×, 2×, and 4× refer to the molar excessof the “bridging” oligonucleotide over the long antisenseoligonucleotide. The concentration of the inhibitor was 10 nM. Threecontrols are: a 22 nt miRNA inhibitor with complete 2′-OMe modification(2′-OMe), miRNA inhibitor X (Exiqon) and miRNA inhibitor Y (Dharmacon).All experiments were performed in triplicate.

FIG. 12 shows the derepression of miR-122 target genes AldoA, Hfe2,S1c35a4, and Lass6 after miR-122 inhibition in mice. mRNA upregulation(%) is normalized to the negative control-injected mice. Three dailyinjections of 50 mg/kg of phosphorothioate (p=s) modified HP#81 andHP#83 miRNA inhibitors were used. All experiments were performed intriplicate.

FIG. 13 shows the potency of the hairpin inhibitors HP#79, HP#85, HP#88,HP#93, and HP#96 targeting let-7a miRNA in Example 9. Potency wasevaluated by quantification of the levels of HMGA2 mRNA expression withTAQMAN® assays, along with three controls: a 22 nt miRNA inhibitor withcomplete 2′-OMe modification (2′-OMe), miRNA inhibitor X (Exiqon) andmiRNA inhibitor Y (Dharmacon). The concentration of miRNA inhibitorsupon transfection: 0.3, 3, 30 nM. Increase of the HMGA2 mRNA levels isshown relative to negative control-transfected samples. All experimentswere performed in triplicate.

FIG. 14 shows the potency of the hairpin inhibitors HP#81, HP#85, HP#88,HP#93, and HP#96 in Example 9. Potency was evaluated using pMIR-REPORT™miRNA expression reporter (miR21 target cloned), along with threecontrols: a 22 nt miRNA inhibitor with complete 2′-OMe modification(2′-OMe), miRNA inhibitor X (Exiqon) and miRNA inhibitor Y (Dharmacon).Concentrations of miRNA inhibitors upon transfection: 30, 3 nM. Theincrease in the firefly luciferase expression induced by miRNAinhibition (the Average fold change) was calculated in the followingway: (fLuc activity anti-miR/βGal activity anti-miR)/(fLuc activityneg/βGal activity neg). All experiments were performed in triplicate.

FIG. 15 shows the potency of the hairpin inhibitors HP#81 andHP#89-HP#92 targeting let-7c miRNA in Example 10. Potency was evaluatedby quantification of the levels of HMGA2 mRNA expression using a TAQMAN®assay. Three controls were used: a 22 nt miRNA inhibitor with complete2′-OMe modification (2′-OMe), miRNA inhibitor X (Exiqon) and miRNAinhibitor Y (Dharmacon). The concentration of miRNA inhibitors upontransfection: 10 nM. All experiments were performed in triplicate.

FIG. 16 shows data derived from the Example 13 experiment in which thepotency of the hairpin inhibitors with a PEG6 vs a PEG5 loop wasevaluated using the pMIR-REPORT™ miRNA expression reporter (miR21 orlet7a target cloned), along with a 22 nt miRNA inhibitor with complete2′-OMe modification (2′-OMe) control. The expression induced by miRNAinhibition is the Average fold change and is calculated in the followingway: (fLuc activity anti-miR/βGal activity anti-miR)/(fLuc activitynegative/βGal activity negative). Note: the higher the bars—the strongerthe miRNA inhibition. The concentration of miRNA inhibitors upontransfection: 0.3 and 3 nM. All experiments were performed intriplicate.

FIG. 17 shows data derived from the Example 14 experiment in which thepotency of miR122-targeting HP#130 inhibitor (with a PEG5 loop) wasevaluated in the exogenous assays. Four constructs were used with clonednatural targets for miR122-RIMS1, GNPDA2, ANKRD13C, and G6PC3 fragments.The expression induced by miRNA inhibition is the Average Fold changeand is calculated in the following way: (fLuc activity anti-miR/βGalactivity anti-miR)/(fLuc activity negative/βGal activity negative). Theconcentration of miRNA inhibitors upon transfection: 3 nM. Allexperiments were performed in triplicate.

FIG. 18 shows data derived from the Example 15 experiment in which thedetection of the hairpin inhibitors with Small RNA TAQMAN® assays wasevaluated. HP#81 inhibitors (with 5′-stem/loop and 2′-OMe modifications)were compared to the unmodified antisense oligonucleotides (unmod),antisense oligonucleotides with complete 2′-OMe modification (2′OMe),inhibitors with 3′-stem/loop and complete 2′-OMe modifications (HP#83),and miRNA inhibitor Y (Dharmacon) featuring both 3′- and 5′-terminalloops and complete 2′-OMe modifications. The miRNA inhibitors in theabove listed formats were synthesized for miR21 and let7c, and detectedwith Small RNA TAQMAN® assays in the cell-free system, in triplicates.

FIG. 19A and FIG. 19B show data derived from the Example 16 experimentin which the specificity of HP#81 inhibitors was studied in theexogenous assays. The inhibitor for miR-21 was tested against itsintended target, miR-21, as well as miR-31, miR-let-7a, miR-106a,miR-23a, miR-19a, miR-17, and miR-24 targets Inhibitor for let-7a wastested against its intended target, miR-let-7a, as well as miR-31,miR-21, miR-106a, miR-23a, miR-19a, miR-17, and miR-24 targets. Theexpression induced by miRNA inhibition is the Average Fold change and iscalculated in the following way: (fLuc activity anti-miR/βGal activityanti-miR)/(fLuc activity negative/βGal activity negative). Theconcentration of miRNA inhibitors upon transfection: 3 nM. Allexperiments were performed in triplicate. FIG. 19A: miR21 target; FIG.19B: let7a target.

FIG. 20A and FIG. 20B show data derived from the Example 17 experimentin which the stability of the hairpin inhibitors HP#81 was studied.Water solutions (20 μmolar) of the inhibitors targeting miR-21 andmiR-23a were stored at +4° C. for 1 week, 2 months and 1 year. Theirpotency was evaluated using the pMIR-REPORT™ miRNA expression reporter(miR21 or miR23a target cloned), along with miRNA inhibitor X (Exiqon).FIG. 20A: miR23a target; FIG. 20B: miR21 target. The expression inducedby miRNA inhibition is the Average fold change and is calculated in thefollowing way: (fLuc activity anti-miR/βGal activity anti-miR)/(fLucactivity negative/βGal activity negative). The concentration of miRNAinhibitors upon transfection: 0.3 and 3 nM. All experiments wereperformed in triplicate.

FIG. 21 shows data derived from the Example 18 experiment in which thederepression (upregulation) of miR-122 target genes AldoA, Hfe2,S1c35a4, and Lass6 after miR-122 inhibition in mice livers was achievedwith the antisense oligonucleotides. Three daily injections of 5 mg/kgof HP#81 and 2′-OMe miRNA inhibitors were used, complexed withINVIVOFECTAMINE® 2.0 reagent. A separate group of mice was injected withmiR122 antagomirs at 50 mg/kg, given in three daily injections. Thenegative control was mice injected with a negative control(non-targeting) miRNA inhibitor, complexed with INVIVOFECTAMINE® 2.0Reagent. mRNA upregulation was normalized to the negative controloligo-injected mice (=100%). Untreated: normal uninjected animals. Allexperiments were performed with 3 animals per group.

FIG. 22 shows a number of exemplary structures of miRNA inhibitors withcholesterol attachments for use in self-delivery applications.

FIG. 23 shows data derived from the Example 17 experiment in which thederepression (upregulation) of miR-122 target genes AldoA, Hfe2,S1c35a4, and Lass6 was achieved after miR-122 inhibition in mice liverswith the antisense oligonucleotides. Three daily injections of 50 mg/kgof HP#115 inhibitors were used. mRNA upregulation was normalized to theuntreated mice (=100%). All experiments were performed with 3 animalsper group.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “siRNA,” and “short interfering RNA” are interchangeable andrefer to unimolecular nucleic acids and to nucleic acids comprised oftwo separate strands that are capable of inducing RNA interference.SiRNA molecules typically have a duplex region that is between 18 and 30base pairs in length.

The term “microRNA”, “miRNA”, and MiR” are interchangeable and refer toendogenous non-coding RNAs that are capable of regulating geneexpression. It is believed that miRNAs function via RNA interference.When used herein in the context of inactivation, the use of the termmicroRNAs is intended to include also long non-coding RNAs, piRNAs,siRNAs, and the like. Endogenous (e.g., naturally occurring) miRNAs aretypically expressed from RNA polymerase II promoters and are generatedfrom a larger transcript.

As used herein “piRNAs” refer to Piwi-interacting RNAs, a class of smallRNAs that are believed to be involved in transcriptional silencing.

As used herein “microRNA inhibitors,” “miRNA inhibitors,” “antisenseinhibitors” “anti-miRs” and “anti-miRNAs” are interchangeable and referto molecules that inhibit the action of miRNAs and the like.

As used herein “alkyl” refers to a hydrocarbyl moiety that can besaturated or unsaturated (alkenyl or alkynyl), and substituted orunsubstituted. It can comprise moieties that are linear, branched,cyclic and/or heterocyclic, and contain functional groups such asethers, ketones, aldehydes, carboxylates, etc. Exemplary alkyl groupsinclude but are not limited to substituted and unsubstituted groups ofmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodcecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl, eicosyl and alkyl groups of highernumber of carbons as well as 2-methylpropyl, 2-methyl-4-ethylbutyl,2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl,6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,and 2-ethylhexyl. The term alkyl also encompasses alkenyl groups, suchas vinyl, allyl, aralkyl, and alkynyl groups.

The term “homologous region” refers to a region of nucleic acid withhomology to another nucleic acid region. Thus, whether a “homologousregion” is present in a nucleic acid molecule is determined withreference to another nucleic acid region in the same or differentmolecule. Further, since nucleic acid is often double-stranded, the term“homologous, region”, as used herein refers to the ability of nucleicacid molecules to be capable of hybridizing to each other. As anexample, a single-stranded nucleic acid molecule can have two homologousregions which are capable of hybridizing to each other. Thus, the term“homologous region” includes nucleic acid segments with complementarysequence. Homologous regions may be of a variety of lengths but willtypically be between 4 and 40 (e.g., from about 4 to about 40, fromabout 5 to about 40, from about 5 to about 35, from about 5 to about 30,from about 5 to about 20, from about 6 to about 30, from about 6 toabout 25, from about 6 to about 15, from about 7 to about 18, from about8 to about 20, from about 8 to about 15, etc.) nucleotides in length.

As used herein “chimeric” refers to a mixture of parts from differentorigins. As used herein, the term can refer to an inhibitor havingcombined parts of the formats shown herein, including at least one ormore of any of parts designated as, but not limited to, BR's, NBR's,S's, HR1 and HR2's, NBR1 and NBR2's, and loops. Further, “chimeric” canrefer to various types of nucleic acids combined into an inhibitor (suchas deoxy-, ribo- or various modified nucleotides). A chimera may alsorefer to combinations of hairpin inhibitors, conjugated antisenseinhibitors and multimeric inhibitors. Another example would be a nucleicacid molecule which contains one region from a mouse chromosome andanother region generated by chemical synthesis and representing anucleotide sequence not known to exist in nature.

The term “sequence identity” refers to the extent to which two sequenceshave the same nucleotide at equivalent positions in a comparison ofnucleic acid usually expressed as a percentage. Thus, two nucleic acidmolecules which differ by one nucleotide over a stretch of 100nucleotides would be said to share 99% sequence identity.

The term “hybridization” refers to the formation of a double-strandednucleic acid (or region) from two single-stranded nucleic acid moleculesor two regions of one nucleic acid molecule.

The term “physiological conditions” refers to environmental conditionswhich are not deleterious to cells such as mammalian cells and includesconditions found, for example, in the cytoplasm of cells. Many of themolecules described herein are designed to function inside cells.Examples of conditions which are not considered to be physiological aretemperatures above 45° C. and pH levels above 9 and below 4. Thus,conditions inside some cellular vacuoles would not necessarily beconsidered to be “physiological”, as that term is used herein.

The term “hairpin” and “stem-loop” can be used interchangeably and referto stem-loop structures. The stem results from two sequences of nucleicacid or modified nucleic acid annealing together to generate a duplex.The loop lies between the two strands comprising the stem.

The term “loop” refers to the part of the stem-loop between the twohomologous regions (the stem) that can loop around to allow base-pairingof the two homologous regions. The loop can be composed of nucleic acid(e.g., DNA or RNA) or non-nucleic acid material(s), referred to hereinas nucleotide or non-nucleotide loops. A non-nucleotide loop can also besituated at the end of a nucleotide molecule with or without a stemstructure.

The term “target” refers to a range of molecules including but notlimited to a miRNA (or pre-miRNAs), an siRNA, a piRNA, a long non-codingRNA, an mRNA, rRNA, tRNA, hnRNA, cDNA, genomic DNA, and long noncodingRNA (ncRNA).

The term “complementary” and “complementarity” are interchangeable andrefer to the ability of polynucleotides to form base pairs with oneanother. Base pairs are typically formed by hydrogen bonds betweennucleotide units in antiparallel polynucleotide strands or regions.Complementary polynucleotide strands or regions can base pair in theWatson-Crick manner (e.g., A to T, A to U, C to G). 100% complementaryrefers to the situation in which each nucleotide unit of onepolynucleotide strand or region can hydrogen bond with each nucleotideunit of a second polynucleotide strand or region. Less than perfectcomplementarity refers to the situation in which some, but not all,nucleotide units of two strands or two regions can hydrogen bond witheach other and can be expressed as a percentage.

As used herein, the “reverse complement” or “RC” refers to a sequencethat will anneal/basepair or substantially anneal/basepair to a secondoligonucleotide according to the rules defined by Watson-Crick basepairing and the antiparallel nature of the DNA-DNA, RNA-RNA, and RNA-DNAdouble helices. Thus, as an example, the reverse complement of the RNAsequence 5′-AAUUUGC-3′ (SEQ ID NO:142) would be 5′GCAAAUU-3′ (SEQ IDNO:143). Alternative base pairing schemes including but not limited toG-U pairing can also be included in reverse complements.

The term “multimeric” refers to a nucleic acid molecule composed ofseveral identical or different subunits held together. As used herein,the term refers to a multimeric nucleic acid molecule made up of severaldifferent sections, some single-stranded, and some double stranded.

As used herein, the term “composite” refers to a structure or an entitymade up of distinct components, such as distinct sections of nucleicacid.

As used herein, the term “nucleotide” or “nt” refers to abase-sugar-phosphate combination. Nucleotides are monomeric units of anucleic acid molecule (DNA and RNA). The term nucleotide includesribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleosidetriphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivativesthereof. Such derivatives include, for example, 2′OMe, 2′ halo such as2′ fluoro or 2′ bromo, LNA, ENA, or 7-deaza such as 7-deaza-dGTP or7-deaza-dATP. The term nucleotide as used herein also refers todideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.Examples of dideoxyribonucleoside triphosphates include, but are notlimited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

As used herein, the phrase “nucleic acid molecule” refers to a sequenceof contiguous nucleotides (riboNTPs, dNTPs or ddNTPs, or combinationsthereof) of any length which can encode a full-length polypeptide or afragment of any length thereof, or which can be non-coding. As usedherein, the terms “nucleic acid molecule” and “polynucleotide” can beused interchangeably and include both RNA and DNA.

As used herein, the term “oligonucleotide” refers to a synthetic ornatural molecule comprising a covalently linked sequence of nucleotideswhich are joined by a phosphodiester bond between the 3′ position of thepentose of one nucleotide and the 5′ position of the pentose of theadjacent nucleotide.

As used herein the term “binding affinity” refers to the affinitybetween two complementary nucleic acid molecules or regions within onenucleic acid molecule. Binding affinity between two complementarynucleic acids will vary with a number of factors including, length, andAT/CG content.

I. Overview

The invention relates to compositions and methods for inactivatingnucleic acid targets. Methods of the invention involve, in part, the useof molecules (e.g., composite inhibitory molecules) with nucleic acidregions with sequence complementarity to the nucleic acid molecule whichis the subject of desired inactivation (e.g., a target nucleic acidmolecule). Compositions described herein can be used for inactivation oftargets that are relatively short non-coding RNAs including regulatoryRNAs. Compositions described herein can be used for the inactivation ofshort non-coding RNAs and long non-coding RNAs. Compositions describedherein can be used for the inactivation of short non-coding RNAs, suchas microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and smallinterfering RNAs (siRNAs). Thus, compositions of the invention caninclude the composite nucleic acid molecules that are microRNAinhibitors (anti-miRs). In some embodiments, compositions of theinvention include at least one miRNA inhibitor and the miRNA inhibitoris produced to target all or a portion of a target nucleic acid. Thoughthey will be referred to as miRNA inhibitors (and anti-miRs), compositenucleic acids can also act to inhibit any target including miRNAs,siRNAs, piRNAs, mRNAs, and rRNAs. Either strand of a duplexed target RNAcan be targeted by miRNA inhibitors provided herein.

In general methods of the invention include the use of nucleic acidmolecules (e.g., composite nucleic acid molecules) that inactivateand/or inhibit miRNAs and other short non-coding RNAs. In someembodiments, methods of the invention involve using inhibitors of threedistinct formats: hairpin inhibitors, conjugated antisense inhibitorsand multimeric inhibitors. Also envisioned are mixtures and chimerathereof. In many instances, all of the formats can be specific for oneor more target nucleic acid molecules (e.g., miRNA).

In some embodiments, target nucleic acid molecules can be obtained froma sample. Samples can be derived from any number of sources. Sincemethods and compositions described herein can be used to inhibit theactivity of miRNAs and the like, most samples will either be known orsuspected to contain one or more target nucleic acid segments (i.e.,miRNAs). Samples can be from viruses, virally infected cells oreukaryotic sources such as protozoa, algae, fungi (yeast and molds),plants, invertebrates (worms), insects (flies), vertebrates, fish,mammals, rodents and primates. Other sources include culture media,either cell free or cell containing as well as cell storage media. Thus,nucleic acid molecules of the invention (e.g., miRNA inhibitors) can beproduced to any target within a sample and/or source.

In some embodiments, mixtures of different nucleic acid molecules of theinvention (e.g., miRNA inhibitors) that target one or more specifictargets can be used. In some embodiments, mixtures of different formatsdisclosed herein can be used to target one or multiple targets (e.g.,one, two, three, four, five, six, seven, eight targets and/or from abouttwo to about fifteen, from about two to about twelve, from about two toabout ten, from about two to about eight, from about two to about six,from about three to about fifteen, from about three to about seven, etc.targets). For example, a mixture including a hairpin inhibitory moleculeand a mixture that includes a conjugated antisense inhibitory moleculecan be used. Mixtures of inhibitors can also include other types ofmiRNA inhibitors known in the art. In some embodiments, a mixture ofvarious hairpin inhibitors (5′ hairpin, 3′ hairpin, and 5′ and 3′hairpin), conjugated antisense inhibitors, and/or multimeric inhibitorscan be used to target a single target. In some embodiments, a mixture ofvarious hairpin inhibitors (5′ hairpin and 3′ hairpin) and/or multimericinhibitors can be used to target multiple targets. In some embodiments,a single miRNA inhibitor can be used that is a chimera of a hairpininhibitor and a multimeric inhibitor, for example, having somemultimeric regions and a 3′ or 5′ hairpin on the same miRNA inhibitorymolecule. In some embodiments, a single miRNA inhibitor can be used formultiple targets. For example, the target binding region can includesequences complementary to two different targets. Alternatively, amultimer can include numerous target binding regions that arecomplementary to one or more targets.

As one skilled in the art would understand, similar embodiments of theinvention may comprise or employ inhibitory molecules which do notinhibit the function of miRNAs. In other words, while reference is madeherein to miRNAs inhibitors, the invention includes inhibitors of otherclasses of nucleic acid molecules. In most instances, inhibitorymolecules will associate a corresponding target by hybridization. Thus,the invention includes methods by which a first molecule (e.g., anucleic acid molecule) binds to a second molecule (e.g., a targetnucleic acid molecule) and inhibits a function (e.g., transcription,translation, RNA interference, etc.) of the second nucleic acidmolecule, as well as compositions of matter used in such methods.

In some embodiments, the miRNA inhibitors, and other inhibitorymolecules of the invention, reduce the amount and/or activity of one ormore targets. In some embodiments, the miRNA inhibitors reduce theamount and/or activity of one or more miRNAs. In some embodiments, themiRNA inhibitors, and other inhibitory molecules of the invention,reduce the amount and/or activity of the target from about 10% to about100%, 20% to about 100%, 30% to about 100%, 40% to about 100%, 50% toabout 100%, 60% to about 100%, 70% to about 100%, 10% to about 90%, 20%to about 85%, 40% to about 84%, 60% to about 90%, including but notlimited to 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%. In some embodiments,the miRNA inhibitors, and other inhibitory molecules of the invention,act to reduce the symptoms of a disease. In some embodiments, the miRNAinhibitors, and other inhibitory molecules of the invention, act tochange a phenotype of a cell, such as a developmental phenotype.

Exemplary inhibitory molecule formats including hairpin inhibitors,multimeric inhibitors, conjugated antisense inhibitors, mixturesthereof, and chimeric inhibitors thereof are explained in more detailwith reference to FIG. 1 and FIG. 9.

TABLE 1A Codes for Formats in Tables 1B-1D Base Code RNA A A RNA C C RNAG G RNA U U DNA A a DNA C c DNA G g DNA T t 2′Ome A A (ital) 2′Ome C C(ital) 2′Ome G G (ital) 2′Ome U U (ital) LNA A A (bold) LNA methyl C C(bold) LNA G G (bold) LNA T T (bold) 2′F C C_(f) 2′F U U_(f)phosphorothioate AC Gt (underlined) C6 amino N 2′O-Propargyl G Y

II. Inhibitory Molecule Formats

Embodiments of inhibitory molecule formats can include hairpininhibitors, conjugated inhibitors, multimeric inhibitors, mixtures andchimera thereof. With reference to FIG. 9, the inhibitors and chimerawill be discussed generally. Then each format will be discussed in moredetail in the following sections A-D.

FIG. 9 shows a schematic of certain formats of the inventions as well asthe inter-relationships of the formats. The formats include a hairpininhibitor (format B1 and C1), a conjugated antisense inhibitor (format Aconjugated to at least one non-nucleic acid moiety, for example), and amultimeric inhibitor (format C2). All of the formats are based on ageneric structure “A” in FIG. 9, including at least one binding region(BR), an optional spacer (S), and at least one non-binding region (NBRor loop). The at least one non-binding region (NBR) may also be anucleotide or non-nucleotide loop and can be at the 5′ and/or 3′ end ofthe binding region (BR) or both the 5′ and 3′ end.

With reference to the generic format “A” in FIG. 9, the binding region(BR) is the region that binds to the target and is also referred to asthe target binding region, the target binding nucleic acid segment, thetarget binding segment and the target binding nucleic acid. The targetcan be any nucleic acid discussed herein as a target, including anyshort, non-coding nucleic acid (e.g., microRNA). The binding region canbe a reverse complement (RC) to one or more target molecule(s) ofinterest (e.g., miRNA). In some embodiments, the length of the targetbinding region is optimized to obtain maximum specificity for the target(e.g., the miRNA) while retaining potency. In some embodiments, thetarget binding nucleic acid segment is complementary to all or a portionof at least one target, such that it will bind to the target (See FIG.9, Target NA in format B1). In some embodiments the portion of thetarget includes at least a part of the 5′ UTR or the 3′ UTR. In someembodiments the portion of the target includes at least a part of acoding region. In some embodiments the target binding nucleic acidsegment is complementary to two or more targets. By complementary, thetarget binding nucleic acid segment can have between about 40% to about100% complementarity with one or more targets, including about 40% toabout 100% complementarity with one or more targets, including about 60%to 100% complementarity with one or more targets, including but notlimited to 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,and 99% complementarity. In some embodiments, the target binding regioncan have 80-100% complementarity with one target and 60% or morecomplementarity with at least a second target. In some embodiments, thetarget binding region can have 100% complementarity to a portion of asingle target and 60% complementarity to the rest of the target. In someembodiments, the target binding nucleic acid segment is at least 6 ormore nucleotides in length. In some embodiments, the target bindingnucleic acid segment is between about 6 and about 200 nucleotides inlength. In some embodiments, the target binding nucleic acid segment isbetween about 6 and 50 nucleotides in length, including between about 9and 25 nucleotides in length, including, but not limited to: 10 nt, 11nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21nt, 22 nt, 23 nt, 24 nt and 25 nt in length. In some embodiments, thetarget binding nucleic acid segment is between about 15 nt and about 25nucleotides in length. In some embodiments, the target binding nucleicacid segment is between about 20 and about 23 nucleotides in length. Thetarget binding region (BR) can be modified in any way known in the artand/or discussed herein (see section entitled “Modification”). In someembodiments, the target binding region is between about 20 and about 200nucleotides in length and the target is a long non-coding RNA.

A spacer (S in FIG. 9) can be used to separate the stem loop, loop orNBR from the target binding nucleic acid segment. The spacer can be oneor more nucleotides. In some embodiments, the spacer is one or morenucleotides having a purine base, a pyrimidine base, or no base (e.g.,abasic). In some embodiments, the spacer is a single nucleotide with thebase A, C, G, U or T. In some embodiments the spacer is at least oneuniversal and/or a modified base. In some embodiments, the spacer is atleast one nucleotide but can be between 0 and 10 nucleotides, including,but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, and 9 nucleotides. In someembodiments, the spacer is one or more nucleotides and can includepurine bases, a pyrimidine bases, and/or no base (e.g., abasic). One ormore nucleotides in the spacer can also be modified (see “Modification”section below). Thus, for example, a conjugated antisense inhibitor caninclude a spacer to separate the binding region (BR) from thenon-nucleotide loop (See A in FIG. 9). For the hairpin inhibitor, thespacer can separate the stem loop from the binding region (see B1 and C1in FIG. 9). For the multimeric inhibitor, the spacer can separate thebinding regions from one or more non-binding regions (see NBR in FIG.9). The spacer can function to reduce or eliminate the charge repulsionof the 5′ end of the NBR or stem (homology region 1) in the respectiveformats for the target nucleic acid. Thus, for example, with referenceto FIG. 9, B1, the 5′ end of the target nucleic acid-bearing phosphatewhen bound to the binding region (BR) may be repulsed. A spacer canreduce this repulsion. The sequence of spacer S1 may be the same as thesequence of spacer S2 or the sequences may be different.

The non-binding region (NBR) in FIG. 9 can be any nucleic acid segmentthat does not bind to the target (a loop, a homology region, a stemloop, or a homology region for the double-stranded part of themultimeric inhibitor). In the case of the conjugated antisenseinhibitor, the NBR can be a non-nucleotide loop (see format A in FIG. 9)and the loop can be attached directly to the inhibitory molecule (withor without a spacer). In the case of the hairpin inhibitor, thenon-binding region can be a stem loop (see format B1 and C1 in FIG. 9).The loop of the stem loop can be a non-nucleotide loop or a nucleotideloop. Alternatively, the NBR (non binding region) can be the part of themultimeric inhibitory molecule that is double-stranded (see NBR1 andNBR2 in FIG. 9, B2A). In this case, while the NBR does not bind to thetarget, it can bind to a reverse complement NBR (NBR2c and NBR1C) tocreate the final multimeric molecule with single stranded regions (BR)and double stranded regions (NBR1 bound to NBR1c and NBR2 bound toNBR2C). A chimeric version of a multimeric inhibitory molecule (see C2in FIG. 9) can also be envisioned to contain one or more loops—eitherstem loops or non-nucleotide loops as non-binding regions.

The loop in the hairpin inhibitor (see “loop” in drawing C1) functionsto connect the two homology regions (HR1 and HR2 in FIG. 9) or stemregions to produce a stem loop. In some embodiments, the inhibitorcontains 2 or more loops. In some embodiments, the 2 or more loops canbe nucleotide loops, non-nucleotide loops and/or mixtures thereof.

When the loop (see drawing “C1”) is composed of nucleic acid, thisnucleic acid can be chemically modified and can be DNA or RNA. Further,the length of the loop can be adjusted to allow for association andbinding between homologous regions (e.g., “HR1” and “HR2” in FIG. 1“C1”). Typically, nucleotide loops will be between about 3 and about 50nucleotides in length (e.g., from about 4 to about 50, from about 6 toabout 50, from about 8 to about 50, from about 10 to about 50, fromabout 4 to about 40, from about 4 to about 30, from about 4 to about 25,from about 4 to about 20, from about 4 to about 15, from about 5 toabout 30, from about 5 to about 15, from about 6 to about 50, from about6 to about 20, from about 8 to about 20, etc. nucleotides).

In some embodiments, the loop (in the hairpin inhibitor) is composed ofnon-nucleotide polymers and derivatives thereof. Briefly, thenon-nucleotide loop can be of sufficient length and/or of sufficientmaterials to enable effective intramolecular hybridization between thehomologous regions (to form a stem). The length of the loop willtypically be a length which is at least the length spanned by at least8-50 atoms (e.g., from about 8 to about 50, from about 8 to about 40,from about 8 to about 30, from about 8 to about 25, from about 8 toabout 20, from about 10 to about 30, from about 12 to about 30, etc.atoms), while not being so long as to interfere with the pairing of theoligonucleotides capable of hybridizing to each other. In someembodiments, the length of the loop is the length spanned by betweenabout 18 and about 22 atoms. In some embodiments, the loop has abackbone of covalently bonded atoms chosen from: carbon, oxygen, sulfur,phosphate, and nitrogen. In some embodiments, the non-nucleotide loophas a backbone composed of covalently bonded carbon atoms. In someembodiments, the backbone of the loop comprises covalently bonded carbonand oxygen atoms. In some embodiments, the loop is chosen frompolyethylene glycol (PEG3-PEG10), C2-C18 alkane diol, styrene, stilbene,triazole, tetrazole, nucleic acid, poly abasic nucleoside,polysaccharide, peptide, polyamide, hydrazone, oxyimine, polyester,disulfide, polyamine, polyether, peptide nucleic acid, cycloalkane,polyalkene, aryl, derivatives thereof and any combination thereof. Insome embodiments, the non-nucleotide loop is a fluorescent dye. In someembodiments, the fluorescent dye is CYANINE™ 5 dye, CYANINE™ 3 dye, oran Alexa Fluor emitter. In some embodiments, the non-nucleotide loop isselected from one of the structures in Table 2. In some embodiments, thenon-nucleotide loop is composed of polyethylene glycol (PEG). In someembodiments, the non-nucleotide loop is composed of a polyethyleneglycol (PEG) derivative. In some embodiments, the non-nucleotide loop iscomposed of a polyethylene glycol (PEG) derivative and the polyethyleneglycol (PEG) derivative is penta-ethylene glycol or hexa-ethyleneglycol. In some embodiments, the non-nucleotide loop is modified byattaching an amine, thiol, NGS ester, alkyne or other functional handlethat could partake in a reaction post synthesis, such as labeling with adye or biotin. Other modifications can be found in references such as:Beaucage, et al, 1992, Tetrahedron, v. 48, p. 2223-2311; Beaucage, etal, 1993, Tetrahedron, v. 49, p. 1925-1963, p6123-6194, and p.10441-10488, herein incorporated by reference. Derivatives ofnon-nucleotide loops can include modified non-nucleotide loops,sidechains, and cycloalkanes addition.

TABLE 2 Non-Nucleotide Loop Structures Link- er Structure Name L1

Triethylene glycol bis- phosphate (TEG) L2

Bis-hexanol disulfide bis-phosphate L3

Hexaethylene glycol bis-phosphate L4

Tetraethylene glycol bis-phosphate L5

Propane diol bis- phosphate L6

Hexane diol bis- phosphate L7

Nonane diol bis- phosphate L8

Dodecyl diol bis- phosphate L10

Bis-propyl-amido stilbene bis-phosphate L11

4-(hydroxybutyl)-1H- 1,2,3-triazol-1-yl)-N-(6- hydroxyhexyl) butanamidebis- phosphate L12

Pentaethylene glycol bis-phosphate

In some embodiments, (the conjugated antisense inhibitor), thenon-nucleotide loop (NBR or loop in format A, FIG. 9) can be anymaterial without limitation and the size is not limiting as long as itdoes not interfere with binding of the binding region (BR) to itstarget. Thus, when the loop is on the 5′ or 3′ end of the conjugatedantisense inhibitor, it can be composed of any of the materials used fora nucleotide loop or for a non-nucleotide loop on the hairpin inhibitor.

In some embodiments, the basic format A inhibitory molecule can alsoinclude a second non-binding region (NBR2) and can be separated from thebinding region (BR) by an optional spacer (S) (see format B2A). Withreference to format B2A, the NBR1 and NBR2 can be any sequence as longas it does not interfere with binding of the target to the bindingregion (BR). Further, the NBR1 and NBR2 can be any sequence as long asNBR1 does not hybridize to NBR2. In some embodiments, the NBR1 and/orNBR2 is between from about 30 nucleotides to about 200 nucleotides inlength, including about 30 to about 100 nucleotides in length, and about30 to about 50 nucleotides in length. In some embodiments, the NBR1and/or NBR2 is from 5 nts to 100 nts in length, or from 5 nts to 50 ntsin length or from 5 to 20 nts in length. The length of NBR1 can be thesame as or different than the length of NBR2.

Inhibitory molecules can also include a second oligonucleotide (see B2Bin FIG. 9) that is capable of binding to the B2A oligonucleotide asfollows. The B2B oligonucleotide can contain a region that is capable ofhybridizing to NBR1 (NBR1C) and a region that is capable of hybridizingto NBR2 (NBR2C). The B2B segment will be the reverse complement of thesesequences and, as such, will be approximately the same length as thesequences. However, the B2B segment can be any length as long as it doesnot interfere with the target binding to the binding region (BR).

With reference to format C2 (the multimeric inhibitor) in FIG. 9, theB2A molecules can be multimerized in a head to tail manner to create thetop strand in the final C2 molecule. Further, the B2B segments can beadded to a mixture containing the B2A molecule and allowed to hybridizesuch that the resulting C2 molecule has some single-stranded regions(the BR with or without the spacer) and some double stranded regions(the B2A segment and the B2B segment). Any of the regions within themultimeric inhibitor (C2) can be modified as discussed in the sectionbelow entitled “Modification”. For example, the binding region can bemodified to enhance binding to its target, and the NBR1 and NBR2 can bemodified to promote binding to the NBR1c and NBR2c regions.Modifications can also be included in any part of the multimericinhibitor, for example, to increase stability or to enhance cell uptake.

In some embodiments, the length of the B2A portion of a multimeric miRNAinhibitor is from about 30 to about 150 nt in length, including about 30to about 100 nt in length, including about 30 to about 45 nucleotides inlength, including, but not limited to, about 31 nt, 32 nt, 33 nt, 34 nt,35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt,and 45 nt. In some embodiments, the length of the B2A portion of amultimeric miRNA inhibitor is from 30 nt, 40 nt, 50 nt, 60 nt, 66 nt, 70nt, 80 nt, 90 nt, 100 nt, or more nucleotides to 175 nt, 200 nt, 250 nt,300 nt, 350 nt, 400 nt, 500 nt, or 660 nucleotides, or any rangetherebetween. For example the length of the miRNA inhibitor may be from30 nt to 250 nt, from 45 to 150 nt, from 50 to 100 nt, from 66 nt to 660nt, from 80 to 500 nt, from 90 to 400 nt, from 100 to 300 nt, from 100to 200 nt, or about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 100 nucleotides.

In some embodiments, a multimeric miRNA inhibitor is made by mixing B2Aand B2B molecules in various molar ratios and allowing the molecules toanneal. Because of this, the length of the final multimeric inhibitorcan vary dramatically, from a single B2A molecule to hundreds of B2Amolecules long. In some embodiments, the multimeric molecules are a poolof various lengths.

In some embodiments, chimeric molecules can be envisioned which containvarious parts of the formats shown herein, including but not limited to,BR's, NBR's, S's, HR1 and HR2's, NBR1 and NBR2's, and loops. Further,the formats can include nucleotide loops or non-nucleotide loops as partof a stem-loop structure or attached at the 3′ or 5′ ends directly. Theformats can also include multimers of any of the above formats and caninclude single and double-stranded regions such as those shown in formatC2. The chimeric molecules of the invention can also include any of themodifications discussed herein and/or under the heading “Modifications”below.

Further, the miRNA inhibitors can include a composition comprisingmixtures of various formats shown in FIG. 1, FIG. 9 and FIG. 10 and/ordiscussed herein.

MiRNA inhibitors can target one or more targets. Thus, each specificbinding region can be complementary to one or more targets.Alternatively, multimers can be made that contain mixtures of B2Amolecules with multiple binding regions each complementary to one ormore targets.

The various formats of the inhibitors shown in FIG. 9 will now bediscussed in more detail under the headings “A. hairpin inhibitors,” “B.conjugated antisense inhibitors,” “C. multimeric inhibitors,” and “D.chimeric inhibitors” below:

A. Hairpin Inhibitory Molecules

Without being limited to a single hypothesis, the hairpin structure actsto “stabilize” one terminus and makes the antisense region moreaccessible for hybridization. Other possibilities are that thedouble-stranded stem region of the hairpin inhibitor (See FIG. 1 and“B1” in FIG. 9) provides an opportunity for a thermodynamicallyfavorable base-stacking interaction between the inhibitory molecule andthe target miRNA. This feature can enhance the ability of inhibitorymolecules to retain the hybridization to the target brought about by thecanonical Watson-Crick complementarity of the strands. However, basestacking is not the only and may not be the major factor. miRNA guidestrands are contained within Ago2 proteins or more sophisticated proteincomplexes of the RNAi machinery. When inhibitors approach the miRNAstrand, hybridization is likely not the major factor; presumably theprotein has a governing role. Its natural target is long and structuredmRNA, so rather than binding the short antisense sequences (even thoughthey are chemically modified and have high melting temperature) theyprefer to bind longer nucleic acids. Hairpin inhibitors seem to work byacting more like the long and structured mRNA with respect to the Agoprotein. Hairpin inhibitors with non-nucleotide loops have the addedadvantage that they are less expensive to produce than those withnucleotide loops, because during oligonucleotide synthesis, one couplingreaction is needed for a non-nucleotide loop whereas nucleotide basedloops need multiple (e.g., 4-5) coupling reactions.

With reference to FIG. 9 B1 and C1, embodiments of hairpin inhibitorsinclude at least one loop (either nucleotide or non-nucleotide loop), afirst homologous region (HR1), a second homologous region (HR2), and atarget binding nucleic acid segment (BR) which is greater than 6nucleotides in length (a composite nucleic acid). The loop (L) functionsto connect the first nucleotide (HR1) and second nucleotide homologousregions (HR2) to form a double-stranded structure (a stem loop) (see C1in FIG. 9). The loop can be composed of nucleotides. Alternatively, theloop can be composed of any type of non-nucleotide material. In eithercase, the loop is of sufficient length to allow for the first and thesecond homologous region to hybridize to one another to form the doublestranded structure (a stem). In some embodiments, the first and secondhomologous regions are able to hybridize under physiological conditions.In some embodiments, the stem-loop is on the 3′ end of the nucleic acidmolecule (3′ hairpin). In some embodiments, the stem-loop is on the 5′end of the nucleic acid molecule (5′ hairpin). In some embodiments, thehairpin inhibitors include at least a second loop (either nucleotide ornon-nucleotide loop), and a second stem region (including a thirdhomologous region, and a fourth homologous region). In some embodiments,the hairpin inhibitor has a single stem-loop region either at the 3′ endor the 5′ end of the molecule. In some embodiments, the stem-loop is onthe 5′ end and there is an addition of at least one nucleotide betweenthe stem and the target binding nucleic acid segment (a spacer). In someembodiments, the stem-loop is on the 3′ end and there is an addition ofat least one nucleotide between the stem and the target binding nucleicacid segment (a spacer). In some embodiments, the hairpin inhibitors areasymmetric structures including a single stem-loop.

When the loop is composed of nucleic acid, this nucleic acid can bechemically modified and can be DNA or RNA. Further, the length of theloop can be adjusted to allow for association and binding betweenhomologous regions (e.g., “HR1” and “HR2” in FIG. 1 and FIG. 9).Typically, nucleotide loops will be between 4 and 50 nucleotides inlength (e.g., from about 4 to about 50, from about 6 to about 50, fromabout 8 to about 50, from about 10 to about 50, from about 4 to about40, from about 4 to about 30, from about 4 to about 25, from about 4 toabout 20, from about 4 to about 15, from about 5 to about 30, from about5 to about 15, from about 6 to about 50, from about 6 to about 20, fromabout 8 to about 20, etc. nucleotides). In some embodiments, the loophas a 2′ position substituted with a molecule chosen from O-alkyl,fluoro, OH and H. Alternatively, the nucleic acid loop can include anyof the modifications discussed herein (see section entitled“Modifications”)

The non-nucleotide loops (see “loop” in FIG. 9, C1) can be used toconnect the homologous nucleic acid segments (HR1 and HR2 in FIG. 9,C1). Briefly, the non-nucleotide loop can be of sufficient length and/orhave a sufficient number of monomers and/or a sufficient number of atomsto enable effective intramolecular hybridization between the homologousregions (to form a stem). In some embodiments, the loop (see “loop” inFIG. 9, C1) is composed of non-nucleotide polymers. The length of theloop will typically be a length which is at least the length spanned byat least 8-50 atoms (e.g., from about 8 to about 50, from about 8 toabout 40, from about 8 to about 30, from about 8 to about 25, from about8 to about 20, from about 10 to about 30, from about 12 to about 30,etc. atoms), while not being so long as to interfere with the pairing ofthe oligonucleotides capable of hybridizing to each other. In someembodiments, the length of the loop is the length spanned by betweenabout 18 and about 22 atoms. In some embodiments, the loop has abackbone of covalently bonded atoms chosen from:

Carbon, Oxygen, sulfur, phosphate, and nitrogen. In some embodiments,the non-nucleotide loop has a backbone composed of covalently bondedcarbon atoms. In some embodiments, the loop is composed of one of themolecules listed in Table 2. In some embodiments, the backbone of theloop comprises covalently bonded carbon and oxygen atoms. In someembodiments, the loop is chosen from polyethylene glycol (e.g., PEG3,PEG4, PEG5, PEG6, PEG7, PEG5, PEG9, or PEG10), C2-C18 alkane diol,styrene, stilbene, triazole, tetrazole, nucleic acid, poly abasicnucleoside, polysaccharide, peptide, polyamide, hydrazone, oxyimine,polyester, disulfide, polyamine, polyether, peptide nucleic acid,cycloalkane, polyalkene, aryl, derivatives thereof and any combinationthereof. In some embodiments, the non-nucleotide loop is a fluorescentdye. In some embodiments, the fluorescent dye is CYANINE™ 5 dye,CYANINE™ 3 dye, or an Alexa Fluor emitter. In some embodiments, thenon-nucleotide loop is composed of polyethylene glycol (PEG). In someembodiments, the non-nucleotide loop is composed of a polyethyleneglycol (PEG) derivative. In some embodiments, the non-nucleotide loop iscomposed of a polyethylene glycol (PEG) derivative and the polyethyleneglycol (PEG) derivative is tri-, tetra-, penta-, hexa-, hepta-, octa-,nona-, or deca-, ethylene glycol ethylene glycol. In some embodiments,the non-nucleotide loop is C12 alkane diol.

In some embodiments, the loop can be separated from the target bindingnucleic acid segment by one or more nucleotide spacers (see FIG. 9,format A, designated as an S). The spacer can be one or morenucleotides. In some embodiments, the spacer is a single nucleotidehaving a purine base, a pyrimidine base, or no base (e.g., abasic). Insome embodiments, the spacer is a single nucleotide with the base A, C,G, U or T. In some embodiments, the spacer is at least one nucleotidebut can be between 0 and 10 nucleotides, including, but not limited to,1, 2, 3, 4, 5, 6, 7, 8, and 9 nucleotides. In some embodiments, thespacer is one or more nucleotides and can include universal bases,purine bases, a pyrimidine bases, and/or no base (e.g., abasic). One ormore nucleotides in the spacer can also be modified (see “Modification”section below). The spacer can function to reduce the possibility thatwhen the target strand binds to the binding region of the inhibitor,there will be charge repulsion between the end of the homologous region(the 3′ phosphate) and the 5′ end of the target.

The first and second homologous regions are of an equivalent length suchthat a stem-like structure can be formed when the two regions bind (seeFIG. 1 and FIG. 9, HR1 and HR2). In some embodiments, the length of thefirst and second homologous regions is between about 3 and 30 nt inlength, including but not limited to 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt,10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt and 30nt in length. In some embodiments, the length of the first and secondhomologous regions is between about 4 and about 10 nucleotides. In someembodiments, the length of the first and second homologous regions isbetween about 4 and about 8 nucleotides. In some embodiments, the firstand second homologous regions are complementary to each other such thatthey can hybridize under physiological conditions. In some embodiments,the first and second homologous regions have between about 40% to about100% complementarity with one or more targets, including about 40% toabout 100% complementarity with one or more targets, including about 60%to 100% complementarity with one or more targets, including 70%, 80%,90%, 95% and 99%. In some embodiments, the first and second homologousregions are not the same length.

The target binding nucleic acid segment shown as “BR” in FIG. 1 and FIG.9, can be a reverse complement (RC) to the target molecule of interest(e.g., miRNA). In some embodiments, the length of the target bindingregion is optimized to obtain maximum specificity for the target (e.g.,the miRNA) while retaining potency. The target can be any nucleic aciddiscussed herein as a target, including any short, non-coding nucleicacid (e.g., microRNA). The binding region can be a reverse complement(RC) to one or more target molecule(s) of interest (e.g., miRNA). Insome embodiments, the target binding nucleic acid segment iscomplementary to all or a portion of at least one target, such that itwill bind to the target (see FIG. 9, Target NA in format B1). Bycomplementary, the target binding nucleic acid segment can have betweenabout 40% to about 100% complementarity with one or more targets,including about 60% to 100% complementarity with one or more targets,including but not limited to 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, and 99% complementarity. In some embodiments thetarget binding region can be complementary to more than one target. Insome embodiments, the binding region can be complementary to two or moretargets. In some embodiments the target binding region is 100%complementary to two or more targets. In some embodiments, the targetbinding region can have 80-100% complementarity with one target and 60%or more complementarity with at least a second target. In someembodiments, the target binding region can have 100% complementarity toa portion of the target and 60% complementarity to the rest. In someembodiments, the target binding nucleic acid segment is at least 6 ormore nucleotides in length. In some embodiments, the target bindingnucleic acid segment is between about 6 and about 200 nucleotides inlength. In some embodiments, the target binding nucleic acid segment isbetween about 6 and 50 nucleotides in length, including between about 9and 25 nucleotides in length, including, but not limited to: 10 nt, 11nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21nt, 22 nt, 23 nt, 24 nt and 25 nt in length. In some embodiments, thetarget binding nucleic acid segment is between about 15 nt and about 25nucleotides in length In some embodiments, the target binding nucleicacid segment is between about 20 nt and about 23 nucleotides in length.The target binding region (BR) can be modified in any way known in theart and/or discussed herein (see section entitled “Modification”). Insome embodiments, the target binding region is between about 20 andabout 200 nucleotides in length and the target is a long non-coding RNA.

In some embodiments, the composite nucleic acid inhibitor (hairpin)molecule can include one or more nucleotides on the 5′ or 3′ end. Insome embodiments, the extra nucleotides can be selected from a purinebase, a pyrimidine base or no base (e.g., abasic). In some embodiments,the extra nucleotides can be from 0 to 30 nucleotides, including but notlimited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 nucleotides. In someembodiments, the composite nucleic acid inhibitory (hairpin) moleculecan include a non-nucleotide loop on the 5′ or 3′ end. Thenon-nucleotide loop can be any material discussed herein as long as itdoes not interfere with binding to the target nucleic acid.

In some embodiments, the total composite nucleic acid inhibitorymolecule (hairpin inhibitor) in total is between about 28 and about 200nucleotides in length, including between about 28 and about 100nucleotides in length. In some embodiments, the composite nucleic acidmolecule is between about 28 and about 40 nucleotides in length. In someembodiments the composite nucleic acid molecule is between about 34 andabout 36 nucleotides in length. In some embodiments, the length does notinclude the non-nucleotide loop.

In some embodiments, the composite nucleic acid molecule (hairpininhibitor) includes modifications in any part of the hairpin inhibitor,including the target binding nucleic acid (“BR” in FIG. 9), the stem(“HR1” and “HR2” in FIG. 9), the loop (“Loop” in FIG. 9), the spacer(“S” in FIG. 9), the 5′ end and the 3′ end. Such modifications can beany modifications that do not preclude binding to the target (e.g., themiRNA). In some embodiments, the modifications increase binding to thetarget. Modifications can include, without limitations, an alkyl, anamine, imine, guanidine, aromatic amino heterocycle. Modifications caninclude DNA, RNA, 2′OMe, 2′OAllyl, 2′O-propargyl, 2′O-alkyl, 2′ fluoro,arabino, 2′-xylo, 2′ fluoro arabino, phosphorothioate,phosphorodithioate, 2′ amino, 5-alkyl-substituted pyrimidine,5-halo-substituted pyrimidine, alkyl-substituted purine,halo-substituted purine, bicyclic nucleotides, 2′MOE, LNA, ENA (e.g.aza-ENA and carbo-ENA), combinations thereof and derivatives thereof.Modifications of these types, as well as other modifications, aredisclosed in US Patent Publ. Nos. 2008/0146788, 2009/0192302, and2010/0087387, the entire disclosures of which are incorporated herein byreference.

Alternatively, modifications can include modifications to thenucleotides in the target binding region, particularly modificationsthat increase binding of the nucleotides to their target. For example,LNA-like molecules are sugar structures that are 3′ endo and are fixedin a certain conformation that is effective to bind to RNA. Exemplarymodifications include at least one 2′ O-alkyl, LNA (and LNA-likemolecules), 2′ fluoro, phosphorothioate, phosphoroamidates, 5-alkyl- orhalo substituted pyrimidines and alkyl or halo-substituted purine bases.Further exemplary modifications include DNA, RNA, 2′OMe, 2′OAllyl,2′O-propargyl, 2′O-alkyl, 2′-xylo, 2′ fluoro, 2′ arabino, 2′ fluoroarabino, phosphorothioate, phosphorodithioate, 2′Amino,5-alkyl-substituted pyrimidine, 5-halo-substituted pyrimidine,alkyl-substituted purine, halo-substituted purine, bicyclic nucleotides,2′MOE, LNA, combinations thereof and modifications thereof. In someembodiments, the modifications can include addition of a 3′ alkyl aminogroup. In some embodiments, the addition of the 3′ alkyl amino groupenhances potency of the microRNA inhibitor at lower concentrations.Exemplary modifications include LNA (and LNA-like molecules) and 2′-OMe,but many other modifications can be included. In some embodiments, thenucleotides are LNA modified at every position. In some embodiments thenucleotides are LNA modified at every other position. In someembodiments, the target binding region is 2′OMe modified. See more aboutmodification in the section entitled “Modifications” below.

With reference to FIG. 1, the hairpin inhibitors can be included in acomposition as 3′ hairpins (having the stem loop at the 3′ end), 5′hairpins (having the stem loop at the 5′ end), 3′ and 5′ double hairpinsand/or combinations of the three. Further, hairpin inhibitors of theinvention can be included in compositions with other types ofinhibitors, including multimeric and/or conjugated antisense inhibitorsof the invention. The asymmetric design can also allow for specificitymodulation. The ability to have two (5′ loop or 3′ loop) highly potentinhibitory molecules for each target miRNA provides not only a means fortarget validation but also a mechanism for studies of specificity withinand among members of closely related miRNA families. 5′ inhibitorsreinforce duplex formation at the 3′ end of the miRNA which cancontribute to inter-familial specificity. Alternatively, 3′ inhibitorsreinforce duplex formation at the 5′ end of the miRNA presumablyreinforcing intra-familial specificity. Thus, mixtures can have a numberof advantages. With reference herein to a 22 nt miRNA inhibitor controlwith complete 2′-OMe modification (2′-OMe), the first 3′-nucleotide forhairpin inhibitors is unmodified as shown in tables and drawings herein.

B. Conjugated Antisense Inhibitors

In some embodiments conjugated antisense inhibitors are envisioned. Withreference to format A in FIG. 9, the conjugated antisense inhibitorsinclude a target binding nucleic acid segment (BR) and at least one loop(NBR or loop), wherein the loop is not nucleic acid. In someembodiments, the loop is located at the 3′ end of the antisenseinhibitor. In some embodiments, the loop is located at the 5′ end of theantisense inhibitor. In some embodiments, the loop is located at bothends of the antisense inhibitor. In some embodiments, when there are twoloops, they are the same non-nucleotide loop. In some embodiments, whenthere are two loops, they are different non-nucleotide loops.

The target binding nucleic acid segment (see “BR” in FIG. 9, format A)can be a reverse complement (RC) to the target molecule of interest(e.g., miRNA). In some embodiments, the length of the target bindingregion is optimized to obtain maximum specificity for the target (e.g.,the miRNA) while retaining potency. The target can be any nucleic aciddiscussed herein as a target, including any short, non-coding nucleicacid (e.g., microRNA). The binding region can be a reverse complement(RC) to one or more target molecule(s) of interest (e.g., miRNA). Insome embodiments, the target binding nucleic acid segment iscomplementary to all or a portion of at least one target, such that itwill bind to the target (see FIG. 9, Target NA in format B1). Bycomplementary, the target binding nucleic acid segment can have betweenabout 40% to about 100% complementarity with one or more targets,including about 60% to 100% complementarity with one or more targets,including but not limited to 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, and 99% complementarity. In some embodiments thetarget binding region can be complementary to more than one target. Insome embodiments, the binding region can be complementary to two or moretargets. In some embodiments the target binding region is 100%complementary to two or more targets. In some embodiments, the targetbinding region can have 80-100% complementarity with one target and 60%or more complementarity with at least a second target. In someembodiments, the target binding region can have 100% complementarity toa portion of the target and 60% complementarity to the rest.

In some embodiments, the target binding nucleic acid segment is at least6 or more nucleotides in length. In some embodiments, the target bindingnucleic acid segment is between about 6 and about 200 nucleotides inlength. In some embodiments, the target binding nucleic acid segment isbetween about 6 and 50 nucleotides in length, including between about 9and 25 nucleotides in length, including, but not limited to: 10 nt, 11nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21nt, 22 nt, 23 nt, 24 nt and 25 nt in length. In some embodiments, thetarget binding nucleic acid segment is between about 15 nt and about 25nucleotides in length In some embodiments, the target binding nucleicacid segment is between about 20 nt and about 23 nucleotides in length.The target binding region (BR) can be modified in any way known in theart and/or discussed herein (see section entitled “Modification”). Insome embodiments, the target binding nucleic acid segment is betweenabout 20 and about 200 nucleotides in length and the target is a longnon-coding RNA.

In some embodiments, the loop (see NBR or Loop in FIG. 9, format A) canbe separated from the target binding nucleic acid segment by a one ormore nucleotide spacer (see FIG. 9, format A, designated as an “S”). Thespacer can be one or more nucleotides. In some embodiments, the spaceris a single nucleotide having a purine base, a pyrimidine base, or nobase (e.g., abasic). In some embodiments, the spacer is a singlenucleotide with the base A, C, G, U or T. In some embodiments, thespacer is at least one nucleotide but can be between 0 and 10nucleotides, including, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, and9 nucleotides. In some embodiments, the spacer is one or morenucleotides and can include universal bases, purine bases, a pyrimidinebases, and/or no base (e.g., abasic). One or more nucleotides in thespacer can also be modified (see “Modification” section below).

In some embodiments, the composite nucleic acid molecule in total(conjugated antisense inhibitor) is between about 15 and about 50nucleotides in length, including between about 20 and about 40nucleotides in length. In some embodiments, the composite nucleic acidmolecule is between about 20 and about 30 nucleotides in length. In someembodiments the composite nucleic acid molecule is between about 20 andabout 25 nucleotides in length. In some embodiments, the length does notinclude the non-nucleotide loop.

In some embodiments, the loop (see “NBR” in FIG. 9, format A) iscomposed of one or more non-nucleotide polymers. In some embodiments,the loop is composed of one of the compounds listed in Table 2. In someembodiments, the non-nucleotide loop has a backbone composed ofcovalently bonded carbon atoms. In some embodiments, the loop has abackbone of covalently bonded atoms chosen from: carbon, oxygen, sulfur,phosphate, and nitrogen. In some embodiments, the backbone of the loopcomprises covalently bonded carbon and oxygen atoms. In someembodiments, the loop is chosen from polyethylene glycol (e.g., PEG3,PEG4, PEG5, PEG6, PEG7, PEG5, PEG9, and PEG10), C2-C18 alkane diol,styrene, stilbene, triazole, tetrazole, nucleic acid, poly abasicnucleoside, polysaccharide, peptide, polyamide, hydrazone, oxyimine,polyester, disulfide, polyamine, polyether, peptide nucleic acid,cycloalkane, polyalkene, aryl derivatives thereof and any combinationthereof. In some embodiments, the non-nucleotide loop is a fluorescentdye. In some embodiments, the fluorescent dye is CYANINE™ 5 dye,CYANINE™ 3 dye, or an Alexa Fluor emitter. In some embodiments, thenon-nucleotide loop is composed of polyethylene glycol (PEG). In someembodiments, the non-nucleotide loop is composed of a polyethyleneglycol (PEG) derivative. In some embodiments, the non-nucleotide loop iscomposed of a polyethylene glycol (PEG) derivative and the polyethyleneglycol (PEG) derivative is a tri-, tetra-, penta-, hexa-, hepta-, octa-,nona-, or deca-, ethylene glycol. In some embodiments, thenon-nucleotide loop is C12 alkane diol.

In some embodiments, the composite nucleic acid inhibitory (conjugatedantisense) molecule can include one or more nucleotides on the 5′ or 3′end. In some embodiments, the extra nucleotides can be selected from apurine base, a pyrimidine base or no base (e.g., abasic). In someembodiments, the extra nucleotides can be from 0 to 30 nucleotides,including but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29nucleotides.

C. Multimeric Inhibitors

Without being limited to a single hypothesis, a multimeric inhibitor canact by mimicking the mRNA and thus, promoting stronger interaction withthe microRNA. Multimeric inhibitors have the further advantage of nothaving secondary structure (in contrast to long single-stranded tandemanti-miRs, which could be chemically synthesized or transcribed invitro) which can complicate complex formation with miRNAs. In addition,the termini of multimeric inhibitors will inherently have extraprotection from RNases, and packaging and in vitro delivery of thesemultimers will be more efficient with many commercially availablereagents because the complexes are long and rigid.

Embodiments of multimeric inhibitors include at least a firstoligonucleotide (e.g., B2A in FIG. 9) and a second oligonucleotide(e.g., B2B in FIG. 9), wherein the first oligonucleotide comprises atarget nucleic acid molecule binding region, and two or more regionswhich will not hybridize to the target nucleic acid molecule, whereinthe second oligonucleotide comprises two regions which will hybridizeunder physiological conditions to at least two of the two or moreregions of the first oligonucleotides which will not hybridize to thetarget nucleic acid molecule, and wherein the multimeric nucleic acidmolecule comprises both single-stranded and double-stranded regions. Insome embodiments, the single-stranded region is capable of binding to atarget nucleic acid molecule under physiological conditions. In someembodiments, the target nucleic acid is a short non-coding RNA. In someembodiments, the target nucleic acid molecule is chosen from a microRNA,a piwi-interacting RNA, a small interfering RNA, a messenger RNA, and aribosomal RNA.

With reference to FIG. 9, an embodiment of a multimeric inhibitorymolecule C2 is shown that is made up of a multimer of molecules B2A withthree molecules of B2B hybridized thereto. Thus, a multimeric inhibitorcan be made up of at least one first oligonucleotide (B2A) thatcomprises a target nucleic acid molecule binding region (BR), and two ormore regions which will not hybridize to the target nucleic acidmolecule (NBR1 and NBR2) and at least one second oligonucleotide (B2B)that comprises two regions which will hybridize under physiologicalconditions (NBR2c and NBR1C) to at least two of the two or more regionsof the first oligonucleotides which will not hybridize to the targetnucleic acid molecule (NBR1 and NBR2), and wherein the multimericnucleic acid molecule (C2) comprises both single-stranded anddouble-stranded regions. Multimeric nucleic acid molecule inhibitors aretherefore comprised of individual molecules that assemble into amultimeric structure. The number of individual molecules may be from 2,3, 4, 5, to 20 or to 100 or more, or any integer range therebetween.Multimeric inhibitors may contain nicks where hybridization of two endsto a complementary nucleic acid occurs.

With reference to FIG. 9 and in more detail, the first oligonucleotideB2A comprises at least one binding region (BR), and at least twonon-binding regions (NBR1 and NBR2). The non-binding regions, while theydo not bind to the target nucleic acid, can bind to other regions of thesecond oligonucleotide (B2B). Thus, the NBR1 is complementary to theNBR1c of the second oligonucleotide (B2B) and the NBR2 is complementaryto the NBR2c of the second oligonucleotide. In some embodiments, thelength of the non-binding regions is between about 3 and 30 nt inlength, including but not limited to 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt,10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt and 30nt in length. In some embodiments, the length of the non-binding regionsis between about 4 and about 10 nucleotides. In some embodiments, thelength of the non-binding regions is between about 4 and about 8nucleotides. In some embodiments, the non-binding regions of the firstoligonucleotide are complementary to the non-binding regions on thesecond oligonucleotide such that they can hybridize under physiologicalconditions. In some embodiments, the non-binding region for the firstoligonucleotide and the non-binding region on the second oligonucleotideare about 80% to about 100% complementary to each other. In someembodiments, the non-binding regions on the first oligonucleotide arecomplementary to the non-binding regions on the second oligonucleotidebut are not the same length.

Although it is not necessary to include spacer regions, in someembodiments, the binding region (BR) of the first oligonucleotide can beseparated from the one or more non-binding regions (NBR1 and/or NBR2) ofthe first oligonucleotide by a one or more nucleotide spacer (see FIG.9, B2A, designated as an “S1” and “S2”). The spacer can be one or morenucleotides. In some embodiments, the spacer is a single nucleotidehaving a purine base, a pyrimidine base, or no base (e.g., abasic). Insome embodiments, the spacer is a single nucleotide with the base A, C,G, U or T. In some embodiments, the spacer is at least one nucleotidebut can be between 0 and 10 nucleotides, including, but not limited to,1, 2, 3, 4, 5, 6, 7, 8, and 9 nucleotides. In some embodiments, thespacer is one or more nucleotides and can include universal bases,purine bases, a pyrimidine bases, and/or no base (e.g., abasic). One ormore nucleotides in the spacer can also be modified (see “Modification”section below). The spacer can function in this inhibitory molecule toensure that there is no charge repulsion from the 3′ phosphate of thesecond oligonucleotide when the target binds to the binding region (BR).

In some embodiments, the binding region binds one or more targets. Thetarget can be any nucleic acid discussed herein as a target, includingany short, non-coding nucleic acid (e.g., microRNA). The binding regioncan be a reverse complement (RC) to one or more target molecule(s) ofinterest (e.g., miRNA). In some embodiments, the length of the targetbinding region is optimized to obtain maximum specificity for the target(e.g., the miRNA) while retaining potency. In some embodiments, thetarget binding nucleic acid segment is complementary to all or a portionof at least one target, such that it will bind to the target (See FIG.9, Target NA in format B1). By complementary, the target binding nucleicacid segment can have between about 40% to about 100% complementaritywith one or more targets, including about 60% to 100% complementaritywith one or more targets, including but not limited to 65%, 70%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% complementarity. Insome embodiments the target binding region can be complementary to morethan one target. In some embodiments, the binding region can becomplementary to two or more targets. In some embodiments the targetbinding region is 100% complementary to two or more targets. In someembodiments, the target binding region can have 80-100% complementaritywith one target and 60% or more complementarity with at least a secondtarget. In some embodiments, the target binding region can have 100%complementarity to a portion of the target and 60% complementarity tothe rest. In some embodiments, the target binding nucleic acid segmentis at least 6 or more nucleotides in length. In some embodiments, thetarget binding nucleic acid segment is between about 9 and 50nucleotides in length, including between about 10 and 25 nucleotides inlength, including, but not limited to: 11 nt, 12 nt, 13 nt, 14 nt, 15nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt and 25nt in length. In some embodiments, the target binding nucleic acidsegment is between about 15 nt and about 25 nucleotides in length Insome embodiments, the target binding nucleic acid segment is betweenabout 20 nt and about 23 nucleotides in length. The target bindingregion (BR) can be modified in any way known in the art and/or discussedherein (see section entitled “Modification”).

In some embodiments, multimers of B2A are linked to form the multimerictop strand of the multimeric inhibitor C2 in FIG. 9. The multimers ofthe B2A molecule include binding regions (BR) separated from otherbinding regions (BR) by one or more non-binding regions (NBR1, NBR2, andNBR1NBR2). The binding regions may also be separated from thenon-binding regions by at least one spacer. Thus, C2 shows one exemplaryformat of a multimeric top strand, but other embodiments can beenvisioned that contain more or fewer B2A molecules and/or spacers. Insome embodiments, some of the B2A molecules linked to create themultimeric top strand in C2 can contain spacers and some may not containspacers. In some embodiments, there are one or more Binding Regions(BR), including but not limited to, two, three, four, five, six, seven,eight, nine, and ten. In some embodiments there are between two and fivebinding regions.

In some embodiments, the first oligonucleotide is between from about 30nucleotides to about 200 nucleotides in length, including about 30 toabout 100 nucleotides in length, and about 30 to about 50 nucleotides inlength.

In some embodiments, the second oligonucleotide is between from about 10nucleotides to about 50 nucleotides in length, including about 10 toabout 30 nucleotides in length, and about 10 to about 20 nucleotides inlength. In some embodiments, the second oligonucleotide does not overlapthe binding region of the first oligonucleotide. In some embodiments,the second oligonucleotide is the same length as the one or morenon-binding regions of the first oligonucleotide that it binds to. It isunderstood that if the first oligonucleotide includes one or more spacerregions that are, for example, 4 nucleotides in length, the secondoligonucleotide could be one or two nucleotides longer (could bindpartially to the spacer region and would not interfere with binding ofthe target molecule.

In some embodiments, because of the way the multimers are formed, thetotal length of the multimeric anti-microRNA molecule (not including thesecond oligonucleotide) can vary from the length of a single B2Amolecule to the length of 100-300 B2A molecules. Thus, depending on theannealing buffer, the number of monomers (B2A and B2B molecules) thatmultimerize can vary. Further, depending on the molar ratio of B2A:B2Bmolecules, the number that multimerize can vary. While the difference insize should not affect the performance, the size can be controlled, ifdesired, by addition of “stoppers/blockers”. The “stoppers/blockers” canbe oligonucleotides that are added with the B2A and B2B molecules duringannealing. The “stoppers/blockers” can contain only one part of thebridging oligo (e.g., only NBR2c without NBR1C). Optionally, a“stopper/blocker” can be a hairpin structure at the 5′ or 3′ end of theB2A molecule. Such blockers could be added to the complexes at differentconcentrations and restrict the average length of the complexes formed.

FIG. 10 shows a schematic of the production of a multimeric inhibitor.With reference to FIG. 10, the top strand is the B2A molecule shown inFIG. 9. The middle strand is the B2B molecule shown in FIG. 9 and thebottom molecule is C2 in FIG. 9. Without being limited to a specifictheory, the single-stranded regions of multimeric inhibitors arebelieved to hybridize to the active miRNA strands loaded onto the Agoprotein. The multimeric inhibitor in FIG. 10 shows two binding regions(the single-stranded regions), but could contain many more depending onhow many B2A molecules multimerize.

In some embodiments, modifications are included that allow for stronginteractions with the target and/or strong interactions between thefirst and second oligonucleotides (B2A and B2B in FIG. 9). Exemplarymodifications include LNA (and LNA-like molecules) and 2′-OMe, but manyother modifications can be included. In some embodiments, thenucleotides are LNA modified at every other position. In someembodiments, the first oligonucleotide and/or the target binding regionis 2′OMe modified.

In some embodiments, the composite nucleic acid inhibitory (multimericinhibitor) molecule can include one or more nucleotides on the 5′ or 3′end. In some embodiments, the extra nucleotides can be selected from apurine base, a pyrimidine base or no base (e.g., abasic). In someembodiments, the extra nucleotides can be from 0 to 30 nucleotides,including but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29nucleotides. In some embodiments, the composite nucleic acid inhibitory(multimeric inhibitor) molecule can include a non-nucleotide loop on the5′ or 3′ end. The non-nucleotide loop can be any material discussedherein as long as it does not interfere with binding to the targetnucleic acid.

In some embodiments, the multimeric molecules are formed by mixingvarious amounts of the long strand containing the target moleculebinding region (the first oligonucleotide) with the smalleroligonucleotide (the second oligonucleotide). In some embodiments, thefirst and second oligonucleotides are mixed in an appropriate buffer(such as an annealing buffer) at a temperature for a time long enough toanneal. In some embodiments, the first and second oligonucleotides aredenatured followed by a cool down for an amount of time to allowannealing of the two molecules. For example, a typical annealing bufferincludes 10 mM Tris pH 7.4, 100 mM NaCl. A typical denaturingtemperature is 95° C. for 3 min. A typical cool down temperature is 37°C. for 1 hour. In some embodiments, the first and secondoligonucleotides are added in equal amounts. In some embodiments, thetwo are added such that there is a molar excess of the firstoligonucleotide. In some embodiments, the two are added such that thereis a molar excess of the second oligonucleotide. In some embodiments,the multimers spontaneously anneal to produce the final multimerizedinhibitory molecules. In some embodiments, the multimers anneal to formmolecules having a variety of sizes (a pool of sizes). In someembodiments, the sizes can be, from a single multimer (the length of oneB2A molecule) to 100s of multimers (100s of B2A molecules in length).

D. Chimera of Inhibitors

In some embodiments, chimeric molecules can be envisioned which containvarious parts of the formats shown herein, including but not limited to,BR's, NBR's, S's, HR1 and HR2's, NBR1 and NBR2's, and loops (see FIG.9). Further, the formats can include nucleotide loop or non-nucleotideloops as part of a stem-loop structure or attached at the 3′ or 5′ endsdirectly. The formats can be produced as multimers and can includesingle and double-stranded regions such as those shown in format C2. Thechimeric molecules of the invention can also include any of themodifications discussed herein and/or under the heading “Modifications”.

In some embodiments, chimeric miRNA inhibitors are produced that haveparts of conjugated antisense inhibitors coupled with parts ofmultimeric inhibitors. For example, parts of the multimeric inhibitorcan be combined with a 3′ or 5′ non-nucleotide loop. Further, chimericinhibitors having parts of the hairpin inhibitors mixed with parts ofthe multimeric inhibitors are envisioned. For example, multimericinhibitors having 5′ or 3′ hairpin structures can be produced and usedfor inhibition of the microRNAs in the cell. In some embodiments,chimeric inhibitors can target one or more targets within a cell orsample.

E. Mixtures of Inhibitors

Compositions of miRNA inhibitors for use in miRNA inhibition can includeany combinations or mixtures of the formats discussed herein. Forexample, combinations can include hairpin inhibitors (with nucleotide ornon-nucleotide loops) mixed with conjugated antisense inhibitors and/ormultimeric inhibitors. Further, combinations can include mixtures ofdifferent types of hairpin inhibitors that are complementary to the sametarget. In some embodiments, mixtures of miRNA inhibitors with a 5′ loop(5′ hairpin) and miRNA inhibitors with a 3′ loop (3′ hairpin) can beused. Having the loop at the 5′ end of the hairpin inhibitors canprovide certain advantages over having the loop at the 3′ end of thehairpin inhibitor (and vice versa). Thus, having a mixture of the twocan be particularly advantageous in some circumstances.

II. Modifications

The composition of the nucleic acid inhibitory molecules of theinvention can vary greatly and can include homogeneous nucleic acids(e.g., all RNA), heterogeneous nucleic acids, (e.g., RNA and DNA),modified nucleic acids, and unmodified nucleic acids. In someembodiments, the nucleic acid molecules of the invention include amixture of modified and unmodified RNA and/or DNA.

Any modification without limitation can be used for the nucleic acidinhibitory molecules of the invention provided they do not inactivatethe inhibitory molecules. The position of the modification can vary withrespect to the following: The position or positions within the strand(i.e., the nucleotide position or positions within the strand orstrands), the positions of the nucleotide(s) that are modified (e.g. thesugar and/or the base), the region of the inhibitory nucleic acid thatis modified (e.g., the target binding region), the number ofmodifications in a specific nucleic acid molecule, and what themodification accomplishes (e.g., increased binding, stability,resistance to RNase, etc.).

In some embodiments, modifications can enhance binding of nucleic acidinhibitory molecules to their target sequences. In some embodiments,modifications can enhance cellular uptake (e.g., endocytosis) of thenucleic acid inhibitor. In some embodiments, modifications can causespecific cellular uptake to a specific cell or tissue. In someembodiments, modifications can enhance cell penetration of the nucleicacid inhibitor. In some embodiments, modifications can enhance cell ortissue localization of the nucleic acid inhibitor. In some embodiments,modifications can enhance facilitated diffusion of the nucleic acidinhibitor. In some embodiments, modifications can enhance cellulartrafficking of the nucleic acid inhibitor. In some embodiments,modifications can enhance binding of the stem structure, thus creating astable stem loop structure. In some embodiments, modifications canreduce the chance of degradation of the nucleic acid inhibitor within acell or tissue. In some embodiments, modifications can reducenon-specific binding of the nucleic acid inhibitor. In some embodiments,modifications can enhance binding of the double stranded regions of amultimeric inhibitor. In some embodiments, modifications can enhanceinhibitor detection of the nucleic acid inhibitor, for example, byadding a tag or detection agent, such as a fluorophore or a radioactivemoiety.

In some embodiments, modifications can be found on any one or moreregions on the nucleic acid inhibitory molecule. With reference to FIG.9, these can include, but are not limited to: a non-binding region(NBR), a binding region (BR), a spacer (S), a homology region (HR), or aloop (nucleotide or non-nucleotide). In some embodiments, modificationsare a 5′ and/or 3′ modification. In some embodiments, modifications arenucleoside modifications. In some embodiments, modifications are basemodifications. In some embodiments, modifications are modifications to anon-nucleotide loop. In some embodiments, modifications are to 1 or morenucleotides within one or more regions within the nucleic acidinhibitory molecule. For example, for a 25 nt binding region, themodification may be to between 1 and 25 nucleotides, including 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,and 24 nucleotides. In some embodiments, modifications are to everyother nucleotide. In some embodiments, every nucleotide in the specifiedregion is modified. For example, in some embodiments, all of thenucleotides of the spacer region (see FIG. 9) are modified. In someembodiments, all of the nucleotides that comprise both the homologousregions and the target binding regions are modified. In someembodiments, all of the nucleotides that comprise both the homologousregions and the target binding regions are 2′OMe modified.

In some embodiments, modifications include the addition of a nucleotideor non-nucleotide loop. In some embodiments, when a nucleotide loop isadded, it is included as a stem-loop structure. In some embodiments, anon-nucleotide loop is composed of one or more of the following:polyethylene glycol (e.g., PEG3, PEG4, PEG5, PEG6, PEG7, PEG5, PEG9, orPEG10), C2-C18 alkane diol, styrene, stilbene, triazole, tetrazole,nucleic acid, poly abasic nucleoside, polysaccharide, peptide,polyamide, hydrazone, oxyimine, polyester, disulfide, polyamine,polyether, peptide nucleic acid, cycloalkane, polyalkene, aryl,derivatives thereof and any combination thereof. In some embodiments,the non-nucleotide loop contains a fluorescent dye. In some embodiments,the fluorescent dye is CYANINE™ 5 dye, CYANINE™ 3 dye, or an Alexa Fluoremitter. In some embodiments, the non-nucleotide loop is composed ofpolyethylene glycol (PEG), fluorescent dye, and a steroid.

In some embodiments, nucleic acid molecules of the invention can includeone or more modifications, including but not limited to: 2′-O-alkylmodifications such as 2′-O-methyl modifications, 2′-orthoestermodifications, 2′ halogen modifications (2′-fluoro), LNAs (lockednucleic acids), LNA derivatives (LNA-like molecules), dithiol, aminoacids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids(e.g., cholesterol) polymers (e.g., PEG), nucleotides, polynucleotides,phosphorothioates, targeted small molecules, fluorescent tags,radioactive labels, derivatives thereof and combinations thereof. Insome embodiments, the modification adds an amine, imine, guanidine, oraromatic amino heterocycle. In some embodiments, nucleic acid moleculescan include modifications chosen from DNA, RNA, 2′OMe, 2′Oallyl,2′O-propargyl, 2′O-alkyl, 2′ fluoro, 2′ arabino, 2′ xylo, 2′fluoroarabino, phosphorothioate, phosphorodithioate, 2′ amino,5-alkyl-substituted pyrimidine, 5-halo-substituted pyrimidine,alkyl-substituted purine, halo-substituted purine, bicyclic nucleotides,2′MOE, LNA, combinations thereof and derivatives thereof.

In some embodiments, the modification comprises at least one or morealkyl groups. Alkyl groups can comprise moieties that are linear,branched, cyclic and/or heterocyclic, and contain functional groups suchas ethers, ketones, aldehydes, carboxylates, etc. Exemplary alkyl groupsinclude but are not limited to substituted and unsubstituted groups ofmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodcecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl, eicosyl and alkyl groups of highernumber of carbons as well as 2-methylpropyl, 2-methyl-4-ethylbutyl,2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl,6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,2-ethylhexyl, and derivatives thereof. The term alkyl also encompassesalkenyl groups, such as vinyl, allyl, aralkyl and alkynyl groups.

Substitutions within alkyl groups, when specified as present, caninclude any atom or group that can be tolerated in the alkyl moiety,including but not limited to halogens, sulfurs, thiols, thioethers,thioesters, amines (primary, secondary, or tertiary), amides, ethers,esters, alcohols and oxygen. The alkyl groups can by way of example alsocomprise modifications such as azo groups, keto groups, aldehyde groups,carboxyl groups, nitro, nitrosos or nitrile groups, heterocycles such asimidazole, hydrazino or hydroxylamino groups, isocyanate or cyanategroups, and sulfur containing groups such as sulfoxide, sulfone,sulfide, and disulfide. Unless otherwise specified, alkyl groups do notcomprise halogens, sulfurs, thiols, thioethers, thioesters, amines,amides, ethers, esters, alcohols, oxygen, derivatives thereof and/or themodifications listed above.

Further, alkyl groups can also contain hetero substitutions, which aresubstitutions of carbon atoms, by for example, nitrogen, oxygen orsulfur. Heterocyclic substitutions refer to alkyl rings having one ormore heteroatoms. Examples of heterocyclic moieties include but are notlimited to morpholine, imidazole, and pyrrolidino.

In some embodiments, modifications can enhance binding to the targetnucleic acid molecule. In some embodiments, such modifications arewithin the target binding region of the inhibitory molecules of theinvention. In some embodiments, the binding of the nucleic acidinhibitor is enhanced as compared to binding of the unmodified nucleicacid inhibitory molecule to its target. In some embodiments, binding isincreased compared to binding to its RNA target. In some embodiments,binding is increased compared to binding to its DNA target. Examples ofmodifications that can enhance the binding of an RNA or DNA to itstarget include but are not limited to: 2′-O-alkyl modifiedribonucleotides, 2′-O-methyl ribonucleotides, 2′-orthoestermodifications (including but not limited to 2′-bis(hydroxyl ethyl), and2′ halogen modifications and locked nucleic acids (LNAs and LNA-likemolecules). However, it is also to be understood that binding can beenhanced by changing the sequence of a region, for example, by includingmore G and C bases. Thus, binding can be increased by increasing the CGcontent of a region. For example, to increase binding of the stem areaof the stem-loop and/or the double-stranded region of the multimericinhibitor, the sequence can be changed to include more cytosines andmore guanines

In some embodiments, modifications can enhance cellular uptake of thenucleic acid inhibitor, specifically or nonspecifically. For example, a3′-terminal cholesterol group appears to aid delivery of inhibitors tocells (Forstemann, et al., 2007, Cell, v. 130, p. 287-297). Further,cholesterol conjugation may also have properties that further enhanceinhibitor activity, such as improved intracellular escape from lipsomes,relocalization of the targeted miRNAs or enhancement of inhibitorstability. Exemplary modifications that can enhance cellular uptakeinclude but are not limited to: 3′Chl_(A) (3′ cholesterol), 3′ Chl_(P)(3′ cholesterol), or phosphorothioates. Antisense inhibitors withcomplete phosphorothioate backbones have been used previously and showno toxicity in mice or primates (Elmén et al, 2008, Nature, v. 452, p.896-900; Elmén et al, 2008, Nucl. Acids Res., v. 36, p. 1153-1162).

The invention thus includes inhibitory molecules which are capable ofcrossing cell membranes. Such molecules will typically be nucleic acidmolecules that are associated with one or more non-nucleic acidmolecules. One example would be a nucleic acid molecule with adouble-stranded region wherein the complementary regions are connectedby a hydrophilic chemical group or collection of groups. In a specificembodiment, a loop region of a miRNA inhibitor may (1) be hydrophilicand/or (2) contain hydrophilic groups and/or regions. For example, theloop may be composed of polyethylene glycol covalently linked to one ormore (e.g., one, two, three, four, five, six, seven, eight, etc.)steroid molecules (e.g., cholesterol, ergosterol, lanosterol, etc., aswell as derivatives thereof). Further, instead of or in addition tosteroid molecules, other hydrophobic molecules may be present (e.g.,lipids such as fatty acyls, sphingolipids, prenol lipids, etc.). Thus,the invention includes methods for delivering inhibitory molecules tocells, as well as the inhibitory molecules themselves. Typically, suchinhibitory molecules will contain a hydrophobic group and/or region.

Additional lipophilic moieties which can be used for delivery ofinhibitory molecules include oleyl, retinyl, cholesteryl residues,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. Additionalcompounds, and methods of use, are set out in US Patent Publication Nos.2010/0076056, 2009/0247608 and 2009/0131360, the entire disclosures ofwhich are incorporated herein by reference.

In some embodiments, modifications can enhance resistance to cellularenzymes, such as RNases and DNases. For example, 2′-O-Meoligonucleotides have been shown to be resistant to cleavage by RNase A,RISC and other cellular ribonucleases (Hutvagner et al., 2004, PLoSBiol. v. 2, p. E98; Meister et al., 2004, RNA, v. 10, p. 544-550).Modifications providing resistance of the nucleic acid inhibitors tonucleases include, but are not limited to, 2′-O-Me, phosphorothioates,LNA (LNA-like molecules), ENA, 2′-MOE, 2′-F, and FANA.

In some embodiments, one or more nucleotide and/or nucleic acidmolecules can be detectably labeled by well known techniques. Detectablelabels include, for example, radioactive isotopes, fluorescent labels,chemiluminescent labels, bioluminescent labels and enzyme labels. Suchlabeled nucleic acid molecules can be used as controls to determineuptake efficiency. In many instances, the detectable label is afluorescent molecule. In some instances, the detectable label is afluorescent (e.g., FITC) or chemiluminescent fluorophore. Fluorescentlabels, nucleic acid molecules, and methods suitable for determining,for example, transfection efficiency are disclosed in US PatentPublication No. 2006/0009409, the entire disclosures of which isincorporated herein by reference.

III. Mode of Action

In some embodiments, inhibitory molecules (e.g., inhibitory nucleic acidmolecules) can act as miRNA inhibitors and/or inactivators in a varietyof ways. Without being bound by any one theory as to why inhibitors ofthe invention perform as inhibitors, they can act by preventingtranscription cleavage and/or by inhibiting translation attenuation. Forexample, a target miRNA can silence its respective target gene byinducing either transcript cleavage (e.g., in cases where the maturemiRNA and target sequence are 100% complementary) or translationattenuation (e.g., in cases where the mature miRNA and target sequenceare less than 100% complementary). Thus, the miRNA inhibitors of theinvention can act by inhibiting either of these actions by the miRNA.

IV. Synthesis of miRNA Inhibitors

The inhibitory molecules (e.g., inhibitory nucleic acid molecules) canbe synthesized by any method that is now known, will be identified inthe future, or is included in this disclosure a person of ordinary skillin the art would appreciate would be useful to synthesize theembodiments taught herein. For example, inhibitory molecules of theinvention can be chemically synthesized using compositions and methodsdescribed in U.S. Pat. Nos. 5,889,136, 6,008,400, 6,111,086, and6,590,093, which are all incorporated by reference herein.

Synthesis methods can include nucleoside base protected5′-O-silyl-2′-O-orthoester-3′-O-phosphoramidites to assemble the desiredunmodified oligonucleotide sequences on a solid support in the 3′ to 5′direction. miRNA inhibitors can be chemically synthesized using standardphosphoramidite-based nucleoside monomers and established solid phaseoligomerization cycles according to Beaucage, S. L. and Iyer, R. P.(Tetrahedron, 1993 (49) 6123; Tetrahedron, 1992 (48) 2223). Purificationof the individual oligonucleotides for in vitro screening can be carriedout using high throughput desalting and alcohol precipitationtechniques. Purification of the individual oligonucleotides for in vivoscreening can be performed with either anion exchange or reverse-phaseprep HPLC and oligonucleotides can be desalted using a semi-permeablemembrane. Analytical HPLC (ion exchange or reverse-phase) can be usedfor determining single strand purity, MALDI mass spectrometry can beused for determining oligonucleotide identity, and UV spectroscopy canbe used for quantitative determination of inhibitors. When a cytidine(C) nucleoside is modified as a bicyclo-sugar, the substitutednucleobase can be a 5′ methylated cytidine residue. When a uridine (U)nucleoside is modified as a bicyclo-sugar, the substituted nucleobasecan be a thymine (T) residue.

In addition, as partially explained above, inhibitory molecules can besynthesized with an array of conjugates for enhancing delivery orallowing visualization of the molecule in a cell or organism. Exemplaryconjugates for delivery include, but are not limited to: cholesterol,folate, PET, peptides, proteins, sugars, carbohydrates, and moieties orcombinations of moieties that enhance cellular uptake. Additionalconjugates can include fluorescent labels, such as fluoroscein,lissamine, phycoerythrin, etc., radioactive labels, or mass labels. Allof the before-mentioned conjugates or labels can be associated with the5′ or 3′ end of the inhibitory molecule or can be conjugated to internalregions.

V. General Methods of Using Inhibitory Molecules/Applications

Inhibitory molecules of the invention can be used for inhibiting orinactivating short non-coding RNAs. In some embodiments, inhibitorymolecules of the invention can be used for inhibiting or inactivatinglong non-coding RNAs, and small non-coding RNAs (e.g., miRNAs, piRNAs,and/or siRNAs) in any cell, tissue, or organism Inhibitory molecules canbe used in basic research, for treatment of a disease, and for modelinga disease. Inhibitory molecules can be used to inhibit miRNA and/orpiRNAs of the human genome implicated in diseases, such as diabetes,Alzheimer's and cancer, and miRNA and/or piRNAs associated with thegenomes of pathogens (e.g., viruses, bacteria, protozoa).

Additionally, inhibitory molecules of the invention can be used in RNAinterference applications, such as diagnostics, prophylactics, andtherapeutics. This can include using inhibitors in the manufacture of amedicament for prevention and/or treatment of animals, such as mammals(e.g., humans). In particular, inhibitory molecules of the invention canbe used to reverse the action of long non-coding RNAs, siRNAs, miRNAs,or piRNAs in disease or therapy.

Inhibitory molecules of the invention can be used in a diverse set ofapplications, including basic research. For example, inhibitorymolecules can be used to validate whether one or more miRNAs or targetsof miRNA can be involved in cell maintenance, cell differentiation,development, or a target for drug discovery or development. Inhibitorymolecules that are specific for inhibiting a particular miRNA areintroduced into a cell or organism and the cell or organism ismaintained under conditions that allow for specific inhibition of thetargeted molecule. The extent of any decreased expression or activity ofthe target is then measured along with the effect of such decreasedexpression or activity, and a determination is made that if expressionor activity is decreased, then the target is an agent for drug discoveryor development. In this manner, phenotyically desirable effects can beassociated with inhibition of particular targets, and in appropriatecases toxicity and pharmacokinetic studies can be undertaken andtherapeutic preparations developed.

Inhibitory molecules of the invention can be used for loss of functionstudies in any cell type or tissue to identify if certain targets areinvolved in disease and/or differentiation.

Inhibitory molecules can be used with diverse cell types, such asprimary cells, germ cell lines, and somatic cells. For example, the celltypes can be embryonic cells, oocytes, sperm cells, adipocytes,fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, bloodcells, megakaryocytes, lymphocytes, macrophages, neutrophyls,eosinophils, basophils, mast cells, leukocytes, granulocytes,keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes andcells of the endocrine or exocrine glands.

Inhibitory molecules of the invention can be used in vivo using methodsand modifications known to those of skill in the art. When used in vivo,inhibitory molecules can contain modifications that aid delivery,uptake, and/or survival intracellularly. Methods of modifying inhibitorymolecules for use in vivo are presented in the section entitled“Modifications.” Further delivery reagents can be used to enhance uptakeand/or delivery of the inhibitors, such as cationic lipids, andlipid-like delivery agents (Semple, et al, 2010, Nature Biotech. Jan.17, p. 1-7; Love et al, 2010, PNAS, v.107, p. 1-6).

Inhibitory molecules can be used as a diagnostic or characterizationtool. For diagnostics, miRNA inhibitors (inhibitory molecules whichblock miRNA function) can be used to treat a sample from a patient thatis believed to have the disease. Based on the cellular consequences tothe sample, the presence of the disease can be determined.Alternatively, miRNA inhibition of a sample of primary cells from apatient can be used to identify the best course of treatment for thespecific patient.

VI. Methods of Using Inhibitory Molecules to Treat Disease

Advantageously, inhibitory molecules can be used to inhibit a broadrange of miRNAs, piRNAs, long non-coding RNAs and siRNAs. For example,inhibitory molecules can be used to inhibit miRNA and/or piRNAs of thehuman genome implicated in diseases, such as diabetes (Poy et al.,Nature 2004, v.432, p. 226-230), Alzheimer's and cancer, and miRNAand/or piRNAs associated with the genomes of pathogens (e.g., pathogenicviruses).

Because a growing body of evidence implicates miRNAs in cancerdevelopment, maintenance and metastasis, it is envisioned thatinhibitory molecules discussed herein can be used to treat cancers. Forexample, Matsuhara et al. (Oncogene 2007, v. 26, p. 6099-6015) foundthat blocking miR-20a and miR-17-5p with antisense oligonucleotidesreduced cell viability and increased the proportion of sub G1 cells.Similarly, Bommer et al. and colleagues (Curr. Biol., 2007, v. 17, p.1290-1307) used propidium iodide stain and FACS to show that miR-34inhibition resulted in increased viability of colon cancer cells. Ma etal. (Nature 2007, v. 449, p. 682-688) found that miR-10b expressionenhanced metastasis; invasive breast cancer cells failed to migrate asfar when treated with a miR-10b antisense oligonucleotide. Antisenseoligonucleotides have also been used in xenograft cancer models todemonstrate that some miRNAs affect metastatic potential and in vivogrowth of tumors. Corsten et al. (Cancer Res. 2007, v. 67, p. 8994-9000)saw that transplanted gliomas treated with miR-21-specific antisenseoligonucleotides were sensitized to a chemotherapeutic agent. Otherstudies showed the involvement of miRNAs in multiple cancer types(Hammond, et al., Can. Chemo. Pharma. 2006, v. 58, s63-s68; Calin etal., Cancer Res 2006, v.66, p. 7390-7394) including leukemia (Calin etal., PNAS 2002, v.101, p. 2999-3004) and glioma (Corsten et al., CancerRes. 2007, v.67, p. 8994-9000).

Further, multiple studies using antisense oligonucleotides suggest rolesfor endogenous miRNAs in viral defense or replication. Thus, it isenvisioned that the miRNA inhibitors discussed herein can be used aloneor in combination with an antiviral drug to treat viral infections. Forexample, Lecellier et al. (Science 2005, v. 308, p. 557-560) found thatantisense oligonucleotide inhibition of miR-32, a miRNA with potentialtarget sites in primate foamy cell virus genes, permitted enhancedproduction of viral RNA in human tissue culture cells. Similarly,inhibition of interferon-β-induced miRNAs permitted Hepatitis C viralproduction. However, antisense oligonucleotide inhibition ofliver-specific miR-122 in cultured hepatocytes crippled Hepatitis Creplication, suggesting its requirement in the Hepatitis C viral lifecycle. These antisense oligonucleotide studies suggest thatcombinatorial expression of pro- or antiviral miRNAs may affect tissuetropism of some viruses.

Inhibitory molecules of the invention can be used to inhibit single ormultiple targets simultaneously. This can be affected by introducingpools of inhibitory molecules targeting different molecules to inhibitdifferent targets. Alternatively, this can be affected by including morethan one target in the target binding region of a single inhibitor.Different types of inhibitors can be pooled together, e.g., (i) miRNAinhibitory molecules of one design (e.g., hairpin) can be pooled withmiRNA inhibitory molecules of another design (e.g., multimer), (ii)miRNA inhibitors with siRNAs, shRNA, or antisense oligos, or ribozymes,and/or deoxyribozymes can be pooled, (iii) inhibitors targetingmicroRNAs and inhibitors targeting other short non-coding RNAs can bepooled Inhibitory molecules of the invention can be used to inhibittargets including but not limited to: miR-20a, miR-17-5p, miR-34,miR-10b, miR-21, miR-32, miR15, miR34a, miR29abc, miR16, let7, miR33,miR221, miR222, miR26a and miR-122. Other disease-related targets can befound via a human miRNA-associated disease database (HMDD), whichcontains miRNA names, disease names, dysfunction, evidence, and PubMedIDs. HMDD is a publicly accessible website (cmbi.bjmu.edu.cn/hmdd).

Inhibitory molecules of the invention can be administered to a cell, byany method known to one of skill in the art. For example, inhibitorymolecules can be passively delivered to cells. Passive uptake can bemodulated, for example, by the presence of a conjugate such as apolyethylene glycol moiety or a cholesterol moiety or any otherhydrophobic moiety associated with the 5′ terminus, the 3′ terminus, orinternal regions of the inhibitor. Other methods for inhibitor deliveryinclude, but are not limited to transfection techniques (e.g., usingforward for reverse transfection techniques) employing DEAE-Dextran,calcium phosphate, cationic lipids/liposomes, microinjection,electroporation, immunoporation, and coupling of the inhibitors tospecific conjugates or ligands such as antibodies, peptides, antigens,or receptors. Other methods include the use of delivery reagents can beenhance uptake and/or delivery of the inhibitors, such as cationiclipids, and lipid-like delivery agents (Semple, et al, 2010, NatureBiotech. Jan. 17, p. 1-'7; Love et al, 2010, PNAS, v.107, p. 1-6). Othermethods include complexing the inhibitor with a lipid.

Inhibitory molecules of the invention can be administered in a dosagethat is therapeutically effective to reduce symptoms of a disease, toreduce the amount and/or symptoms of a pathogen, and or to reduce theamount of siRNAs, long non-coding RNAs, miRNAs, and/or piRNAs that arebeing targeted. The dosages can vary from micrograms per kilogram tohundreds of milligrams per kilogram of a subject. As is known in theart, dosage will vary according to the mass of the mammal receiving thedose, the nature of the mammal receiving the dose, the severity of thedisease or disorder, and the stability of the medicament in the serum ofthe subject, among other factors well known to persons of ordinary skillin the art. Results of the treatment can be ameliorative, palliative,prophylactic, and/or diagnostic of a particular disease or disorder.Suitable dosing regimens can be determined by, for example,administering varying amounts of one or more inhibitors in apharmaceutically acceptable carrier or diluents by a pharmaceuticallyacceptable delivery route, and the amount of drug accumulated in thebody of the recipient organism can be determined at various timesfollowing administration. Similarly the desired effect can be measuredat various times following administration of the inhibitor and this datacan be correlated with other pharmacokinetic data such as body or organaccumulation. Those of ordinary skill can determine optimum dosages,dosing regimens, and the like. The inhibitors can be administered incombination with other pharmaceuticals for treatment of a disease. Insome embodiments, inhibitory molecules can be administered to a patientthat has been exposed to a disease and/or to keep a patient from gettinga disease they will be exposed to (proactively).

Inhibitory molecules can be administered using any route ofadministration known to one of skill in the art. For example, inhibitorymolecules can be administered in a cream or ointment topically, an oralpreparation such as a capsule or tablet or suspension or solution, andthe like. The route of administration can be intravenous intramuscular,dermal, subdermal, cutaneous, subcutaneous, intranasal, oral, rectal, byeye drops, by tissue implantation of a device that releases theinhibitor at an advantageous location, such as near an organ or tissueor cell type harboring a target nucleic acid of interest.

VII. Quantifying Inhibitory Molecule Function

The method of assessing the level of inhibition or inactivation is notlimited. Thus, the effects of any inhibitory molecules can be studied byone of any number of procedures. Methods include, but are not limitedto, the biological assay 1 or 2 herein (Examples 1-6), Northernanalysis, RT PCR, expression profiling, and others. In some methods, avector or plasmid encoding a reporter whose protein is easily assayed isused. The vector or plasmid is modified to contain the target site(e.g., the reverse complement of the mature long non-coding RNA, miRNA,piRNA, or siRNA) in the 5′ UTR, ORF, or 3′UR of the sequence. Suchreporter genes include but are not limited to alkaline phosphatase (AP),beta galactosidease (LacZ), chloramphenicol acetyltransferase (CAT),green fluorescent protein (GFP), variants of luciferase (Luc), andderiviatives thereof. In the absence of inhibitory molecules, endogenous(or exogenously added) miRNAs target the reporter mRNA for silencing(e.g., either by transcript cleavage or translation attenuation) thusleading to an overall low level of reporter expression. In contrast, inthe presence of inhibitory molecules of the invention, miRNA, piRNA, orsiRNA mediated targeting is suppressed, thus giving rise to a heightenedlevel of reporter expression.

VIII. Self-Delivery Aspect of the Molecules

The invention also provides molecules which are capable of“self-delivery”. “Self-delivery” refers to the ability of a molecule toefficiently cross a cell membrane in the absence of the addition of atransfection reagent (e.g., 293FECTINT™, OPTIFECT™, LIPOFECTIN®,LIPOFECTAMINE® 2000, etc.). “Self-delivery” may also be measured basedupon a functional effect resulting from entry into a cell by themolecule (e.g., induction of apoptosis). Self-delivery may be in vitroor in vivo. Examples of in vitro self-delivery include instances wherecells in a well of a microtiter or in a culture medium flask are exposedto molecules which efficiently cross the cell membrane in the absence ofa transfection reagent.

As one skilled in the art would understand, it is generally notdesirable to introduce molecules (e.g., nucleic acid molecules of theinvention) into multicellular organisms (e.g., animals) in conjunctionwith a transfection reagent. Further, it is generally desirable tointroduce the fewest possible number of compound, for example, into theblood stream of an animal required to achieve the desired goal (e.g.,inhibition of miRNA function). Thus, the invention provides moleculeswhich are designed to allow for in vivo self-delivery.

In many instances, molecules which are capable of self-delivery willcontain a hydrophobic region, hydrophobic chemical entity or set ofhydrophobic chemical entities. Further, when present, hydrophobicregions or hydrophobic chemical entities may be located anywhere on themolecules and may be attached to the molecules either covalently ornon-covalently.

With respect to molecules of the type shown in FIG. 1 for reference,hydrophobic regions or hydrophobic chemical entities may be located, forexamples, on the 3′ terminus (attached to a hydroxyl group or aphosphate group) and/or 5′ terminus (attached to a hydroxyl group or aphosphate group). Further, hydrophobic regions or hydrophobic chemicalentities may be located in one or more loop region or attached to thesugar backbone of a stem (e.g., any one, two, three or all four stemregions) or the binding region. Thus, as an example, the inventionincludes molecules which contain hydrophobic regions or hydrophobicchemical entities located at the 3′ terminus, loop 1, and loop 2.

The number of hydrophobic regions or hydrophobic chemical entitiesassociated with each molecule capable of self-delivery can vary widelybased upon factors such as the desired hydrophobic character of thefinal molecule and locations which contain the hydrophobic regions orentities. In specific embodiments, from about one to about fifty (e.g.,from about one to about forty, from about one to about thirty, fromabout one to about twenty, from about one to about ten, from about twoto about fifteen, from about two to about five, from about three toabout eight, from about four to about ten, etc.) hydrophobic regions orhydrophobic chemical entities may be present on each molecule capable ofself-delivery. These numbers may refer to averages of hydrophobicregions or hydrophobic chemical entities present on each molecule. Thisis so because molecules in a population may vary in the presence offunctional groups as a result of factors such as incomplete chemicalmodification or post chemical modifcation reactions. In most instances,greater than 50% of the molecules in the population will havehydrophobic regions or hydrophobic chemical entities in all of thedesired locations.

Hydrophobic regions or hydrophobic chemical entities may be eitherdirectly or indirectly (e.g., through a linker) linked to a group of anucleic acid molecule. (For example, in example 7 multimeric inhibitorsare described. Sterols or any other hydrophobic groups can be attachedfor example to the bridging, non-functional oligonucleotide, while thefunctional oligonucleotide with the antisense region-targetingmiRNA-could be unmodified) One example of such a group is the 2′ OH ofribose. Further, multiple (e.g., from about two to about twenty, fromabout two to about fifteen, from about two to about ten, from about twoto about five, from about two to about three, from about three to aboutten, from about four to about ten, etc.) groups of a nucleic acidmolecule may contain the same or different hydrophobic regions orhydrophobic chemical entities, some, all or none or which may beconnected to the nucleic acid molecule through one or more linkers.Also, a specified percentage (e.g., from about 10% to about 100%, fromabout 20% to about 100%, from about 30% to about 100%, from about 40% toabout 100%, from about 50% to about 100%, from about 10% to about 20%,from about 10% to about 30%, from about 10% to about 40%, from about 10%to about 50%, etc.) of the nucleosides present may contain the same ordifferent hydrophobic regions or hydrophobic chemical entities.

Hydrophobic chemical entities employed in the practice of the inventioninclude lipids (e.g., steroids). Exemplary lipids can be simple lipids(esters of fatty acids with various alcohols); complex lipids such asphospholipids and glycolipids; derived lipids such as fatty acids,higher alcohols, lipid soluble vitamins, steroids, and hydrocarbons. Toenhance the cellular uptake efficiency, the lipid may be a derivedlipid, such as a fatty acid having from about 6 to about 50 (e.g., fromabout 10 to about 22, from about 12 to about 18, from about 6 to about22, from about 6 to about 40, from about 10 to about 40, from about 20to about 40, from about 20 to about 50, etc.) carbon atoms. Specificfatty acids which may be used in the practice of the invention includelauric acid, stearic acid, myristic acid, and palmitic acid. Inaddition, lipid conjugates such as cardiolipin, ceramides, andsphingolipids which can be attached to oligonucleotides.

Any number of different steroids may be used in the practice of theinvention. Exemplary steroids include cholesterol, acebrochol,androsterone, 5-beta-cholanic acid, progesterone, aldosterone,dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol,dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone,testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol,corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon,equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol,mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene,fusidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol,dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone,hydrocortisone, parametasone, betametasone, cortisone, fluocinonide,fluorometholone, halcinonide, and budesonide, Calciferol,Cholecalciferol, Deoxycholic acid. Cholic acid, Hydrodeoxycholic acid,Lithocholic acid, Ursodeoxycholic acid, Prednisone. Dehydrocholic acid,as well as any of these molecules further substituted with one or morehydroxyl, halogen, amino, alkylamino, alkyl, carboxylic acid, ester,amide, carbonyl, alkoxyl, or cyano groups. FIG. 22 provides a number ofstructures depicting attachment of cholesterol to the miRNAs of theinvention.

Hydrophobic chemical entities employed in the practice of the inventioninclude hydrocarbons with alkyl groups. As used herein, the term “alkyl”refers to a hydrocarbyl moiety that can be saturated or unsaturated, andsubstituted or unsubstituted. It may comprise moieties that are linear,branched, cyclic and/or heterocyclic, and contain functional groups suchas ethers, ketones, aldehydes, carboxylates, etc. Exemplary alkyl groupsinclude but are not limited to substituted and unsubstituted groups ofmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl, eicosyl and alkyl groups of highernumber of carbons, as well as 2-methylpropyl, 2-methyl-4-ethylbutyl,2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl,6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,and 2-ethylhexyl. The term alkyl also encompasses alkenyl groups, suchas vinyl, allyl, aralkyl and alkynyl groups. The alkynyl groups may besubstituted.

Substitutions within alkyl groups, when specified as present, caninclude any atom or group that can be tolerated in the alkyl moiety,including but not limited to halogens, sulfurs, thiols, thioethers,thioesters, amines (primary, secondary, or tertiary), amides, ethers,esters, alcohols and oxygen. The substitutions within alkyl groups can,by way of example, also comprise modifications such as azo groups, ketogroups, aldehyde groups, carboxyl groups, nitro, nitroso or nitrilegroups, heterocycles such as imidazole, hydrazino or hydroxylaminogroups, isocyanate or cyanate groups, and sulfur containing groups suchas sulfoxide, sulfone, sulfide, and disulfide. Alkyl groups may containhalogens, sulfurs, thiols, thioethers, thioesters, amines, amides,ethers, esters, alcohols, oxygen, or the modifications listed above.

Further, alkyl groups may also contain hetero substitutions, which aresubstitutions of carbon atoms, by, for example, nitrogen, oxygen orsulfur. Heterocyclic substitutions refer to alkyl rings having one ormore heteroatoms. Examples of heterocyclic moieties include but are notlimited to morpholino, imidazole, and pyrrolidino. Alkyl groups maycontain hetero substitutions or alkyl rings with one or more heteroatoms(i.e., heterocyclic substitutions).

Nucleic acid molecules with enhanced capability of crossing cellmembranes in the absence of a transfection reagent are set out in U.S.Pat. Publ. 2008/0085869A1, the entire content of which is incorporatedherein by reference.

Unmodified nucleic acid molecules have some ability to cross cellmembranes. Thus, self-delivery refers to an increased ability to entercells. Uptake can be measured from the quantitative perspective of thetotal amount of a molecule taken up or the qualitative perspective offunctional activity associated with uptake. Qualitative uptake may notexactly correlate with quantitative uptake because the functionalactivities of molecules capable of self-delivery may vary from those notcapable of self-delivery. Functional activities of two molecules can becompared by measurement of (1) total uptake of each molecule and (2)resulting activity. By such methods one can determine quantitativeuptake from functional activity.

Regardless of how uptake is measured, the invention provides nucleicacid molecules capable of self-delivery. Nucleic acid molecules capableof self-delivery may be taken up by cells more than 40% (e.g., fromabout 40% to about 90%, from about 50% to about 90%, from about 60% toabout 90%, from about 70% to about 90%, from about 75% to about 90%,from about 50% to about 98%, from about 60% to about 99%, from about 70%to about 99%, etc.) more efficiently than unmodified nucleic acidmolecules (e.g., nucleic acid molecules represented in FIG. 1). In oneembodiment, the invention provides nucleic acid molecules capable ofself-delivery which are taken up at least 75% more efficiently thanotherwise identical unmodified nucleic acid molecules. Quantification ofuptake will generally be determined by quantification of the numbers ofindividual molecules (e.g., moles) present in cells.

The invention provides, in part, molecules which may be administered toa multicellular organism (e.g., a plant or animal) to achieve a specificintracellular effect (e.g., the inhibition of the function of a specificmiRNA, such as MiR-122). As shown in Example 19, sterol conjugatedmolecules of the invention can be administered to animals, with themeasurement of effect in a distant organ (i.e., liver in this instance).Thus, the invention provides methods for the systemic and organ specificdelivery of molecules of the invention. The invention further providesmethods for the treatment of disease.

The invention thus includes methods for delivering nucleic acidinhibitory molecules of the invention to cells of a multicellularorganism. Such delivery may be local or systemic.

Local delivery of nucleic acid molecules (e.g., nucleic acid moleculesof the invention) is well suited for afflictions where administrationsites are easily accessible. Examples are such administration sites areskin and mucosal surfaces. Local delivery has the advantage of lesseningpotential side effects resulting from systemic administration. Further,local administration avoids first-pass hepatic clearance making it morelikely that the therapeutic concentration is reached at the target sitein sufficient concentrations to achieve the desired effect. Localdelivery can be generally categorized into five main groups: mucosal(intranasal, intratracheal, intravaginal and intrarectal), intraocular,transdermal, intrathecal and intratumoral, any of which may be used inthe practice of the invention. Local delivery also has the advantagethat transfection reagents can often be readily used with limited effecton multicellular organisms. Thus, self-delivering andnon-self-delivering may be easily used.

Systemic administration may be used to deliver nucleic acid molecules(e.g., nucleic acid molecules of the invention). In many instances, suchdelivery would be for the treatment of diseases (e.g., cancer, metabolicdisorders, etc.) where the target sites are not easily accessible tolocal administration. Systemic administration can be performed through,for examples, intravenous, intraperitoneal, intramuscular, orsubcutaneous injections. Intravenous administration, as an example, isthe widely used, simple with respect to procedure, and results in rapiddistribution of administered agent to various tissue sites.

The invention thus includes methods for administering and methodinvolving administration of molecules of the invention to multicellularorganisms (e.g., animals), as well as composition used in suchadministration. In some embodiments, such methods include method oftreating afflictions/diseases.

The invention further includes pharmaceutical compositions comprisingmolecules of the invention, as well as admixtures comprising moleculesof the invention and additional compounds for administration tomulticellular organisms (e.g., buffers, excipients, etc).

In some embodiments, the invention includes compositions comprising (1)one or more molecules of the invention and (2) an excipient formulation,as well as methods for preparing such compositions and methods foradministering such compounds to multicellular organisms. Exemplaryexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In some embodiments, molecules of the invention as delivered tomulticellular organisms as components of compositions including, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inspecific embodiments, a pharmaceutically acceptable carrier is used suchas a liposome or a transdermal enhancer.

Exemplary delivery systems of the invention include patches, tablets,suppositories, pessaries, gels and creams, and can contain excipientssuch as solubilizers and enhancers (e.g., propylene glycol, bile saltsand amino acids), and other vehicles (e.g., polyethylene glycol, fattyacid esters and derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Molecules of the invention may be formulated or complexed withpolyethylenimine (e.g., linear or branched PEI) and/or polyethyleniminederivatives, including for example grafted PEIs such as galactose PEI,cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI(PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPAPharm. Sci., 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14,840-847; Kunath et al., 2002, Pharmaceutical Research, 19, 810-817; Choiet al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al.,1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002,Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of GeneMedicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA, 96,5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60,149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, the entire disclosure of which isincorporated by reference herein.

Molecule of the invention comprises a bioconjugate, for example anucleic acid conjugate as described in Vargeese et al., U.S. PatentPublication No. 2003/0130186 A1, U.S. Pat. No. 6,528,631; U.S. Pat. No.6,335,434; U.S. Pat. No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat.No. 5,214,136; U.S. Pat. No. 5,138,045, the entire disclosures of whichare incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more molecules of the invention in an acceptable carrier, such as astabilizer, buffer, and the like. Molecules of the invention can beadministered and introduced to a subject by any standard means, with orwithout stabilizers, buffers, and the like, to form a pharmaceuticalcomposition. When it is desired to use a liposome delivery mechanism,standard protocols for formation of liposomes can be followed. Thecompositions of the present invention can also be formulated and used ascreams, gels, sprays, oils and other suitable compositions for topical,dermal, or transdermal administration as is known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

As used herein, a pharmacological composition or formulation includescompositions or formulations in forms suitable for administration, e.g.,systemic or local administration, into a cell or subject, including forexample a human. Suitable forms, in part, depend upon the use or theroute of entry, for example oral, transdermal, or by injection. Suchforms should not prevent the composition or formulation from reaching atarget cell (i.e., a cell to which the negatively charged nucleic acidis desirable for delivery). For example, pharmacological compositionsinjected into the blood stream should be soluble. Other factors areknown in the art, and include considerations such as toxicity and formsthat prevent the composition or formulation from exerting its effect.

Molecules of the invention may be administered to subjects by systemicadministration in a pharmaceutically acceptable composition orformulation. “Systemic administration” includes in vivo systemicabsorption or accumulation of drugs in the blood stream followed bydistribution throughout the entire body. Administration routes that leadto systemic absorption include, without limitation: intravenous,subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary andintramuscular. Each of these administration routes may be used to exposemolecules of the invention to an accessible diseased tissue. The use ofa liposome or other drug carrier comprising the compounds of the instantinvention can potentially localize molecules of the invention, forexample, in certain tissue types, such as the tissues of the reticularendothelial system (RES). In some instances, liposome formulations thatcan facilitate the association of molecules of the invention with thesurface of cells, such as, lymphocytes and macrophages may also beuseful. This approach can provide enhanced delivery of molecules of theinvention to target cells by taking advantage of the specificity ofmacrophage and lymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of molecules of the invention inthe physical location most suitable for their desired activity.Non-limiting examples of agents suitable for formulation with themolecules of the invention include: P-glycoprotein inhibitors (such asPluronic P85); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery(Emerich, D F et al., 1999, Cell Transplant, 8, 47-58); and loadednanoparticles, such as those made of polybutylcyanoacrylate. Othernon-limiting examples of delivery strategies for molecules of theinvention include material described in Boado et al., 1998, J. Pharm.Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284;Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. DrugDelivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic AcidsRes., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al., Chem. Rev.1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43,1005-1011). Such liposomes have been shown to accumulate selectively intumors, presumably by extravasation and capture in the neovascularizedtarget tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulatingliposomes enhance the pharmacokinetics and pharmacodynamics of DNA andRNA, particularly compared to conventional cationic liposomes which areknown to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem.1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat of a disease state (e.g., alleviate asymptom or inhibit a disease state mechanism). The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.01 mg/kg and 300 mg/kgbody weight/day (e.g., from about 0.1 mg/kg to about 100 mg/kg, fromabout 0.5 mg/kg to about 100 mg/kg, from about 1 mg/kg to about 100mg/kg, from about 5 mg/kg to about 100 mg/kg, from about 0.01 mg/kg toabout 3 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, from about 0.01mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 0.1 mg/kg, fromabout 3 mg/kg to about 20 mg/kg, from about 2 mg/kg to about 40 mg/kg,from about 5 mg/kg to about 50 mg/kg, etc.) of active ingredients isadministered to achieve the desired physiological effect (e.g.,inhibition of microRNA activity).

The amount of a molecule of the invention administered to amulticellular organism will vary with the desired and/or requiredeffect. For example, treatment of an affliction may require at least 70%inhibition of a microRNA in a specific tissue (e.g., prostate glandtissue). Thus, the invention includes methods for using molecules of theinvention which yield a desired effect. The effect can be empirical(remission of a diseases state). The effect can also be measured throughthe measurement of the activity of one or more molecules targeted bymolecules of the invention (e.g., microRNAs). In such instances, theinvention provides methods for inhibition of target molecule activitywithin a multicellular organism where activity of one or more targetmolecules is inhibited by at least 30% (e.g., from about 30% to about99%, from about 40% to about 99%, from about 50% to about 99%, fromabout 60% to about 99%, from about 70% to about 99%, from about 80% toabout 99%, from about 30% to about 95%, from about 40% to about 95%,from about 60% to about 95%, from about 30% to about 90%, from about 40%to about 90%, from about 50% to about 90%, from about 60% to about 90%,from about 30% to about 85%, from about 40% to about 85%, from about 50%to about 85%, from about 60% to about 85%, from about 70% to about 90%,etc.).

Molecules of the invention and formulations thereof can be administeredorally, topically, parenterally, by inhalation or spray, or rectally indosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and/or vehicles. Theterm parenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, or intrathecalinjection or infusion techniques and the like. In addition, there isprovided a pharmaceutical formulation comprising one or more molecule ofthe invention and a pharmaceutically acceptable carrier. One or moremolecules of the invention can be present in association with one ormore non-toxic pharmaceutically acceptable carriers and/or diluentsand/or adjuvants, and if desired other active ingredients.Pharmaceutical compositions containing molecules of the invention can bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsion, hard or soft capsules, or syrups or elixirs.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.Aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. Oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Pharmaceutical compositions can be in the form of a sterile injectableaqueous or oleaginous suspension. Such suspensions can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents that have been mentioned above. Sterileinjectable preparation can also be a sterile injectable solution orsuspension in a non-toxic parentally acceptable diluent or solvent, forexample as a solution in 1,3-butanediol. Among the acceptable vehiclesand solvents that can be employed are water, Ringer's solution andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids, such as oleic acid, may beused in the preparation of injectables.

Molecules of the invention can also be administered in the form ofsuppositories, e.g., for rectal administration of the drug. Suchcompositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Molecules of the invention can be administered parenterally in a sterilemedium. Molecules of the invention, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

For administration to non-human animals, molecules of the invention canalso be added to the animal feed or drinking water. It can be convenientto formulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

Molecules of the invention can also be administered to a subject incombination with other therapeutic compounds to increase the overalltherapeutic effect. In some instances, the use of multiple compounds totreat an indication can increase the beneficial effects while reducingthe presence of side effects.

The invention also comprises compositions suitable for administeringmolecules of the invention to specific cell types. For example, theasialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem.262, 4429-4432) is unique to hepatocytes and binds branchedgalactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Inanother example, the folate receptor is overexpressed in many cancercells. Binding of such glycoproteins, synthetic glycoconjugates, orfolates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Patent PublicationNo. 2003/0130186 A1 and Matulic-Adamic et al., U.S. Pat. No. 7,833,992,the entire disclosure of which is incorporated by reference herein.

IX. Kits

Some embodiments of the invention provide kits for the inhibition of oneor more target nucleic acid molecules (i.e., miRNAs) in a sample,including at least one hairpin inhibitor, at least one conjugatedantisense inhibitory molecule, at least one multimeric inhibitorymolecule, chimeric inhibitory molecules, and/or mixtures thereof. Thekit can be used in any of the methods of using the miRNA inhibitorymolecules known or disclosed herein. Some embodiments of the kit areused for inhibition of a long non-coding RNA, an miRNA, a piRNA, ansiRNA, an mRNA, and/or an rRNA in a cell or organism. In someembodiments the kit is used to identify a treatment for a disease or toproduce a treatment for a disease in a mammal.

Having described the invention with a degree of particularity, exampleswill now be provided. The following examples are intended to illustratebut not limit the invention.

EXAMPLES

The sequences and formats of the inhibitory molecules used in thefollowing examples are presented in Tables 1A-1D. The codes for themodifications are shown in Table 1A. Briefly, RNA is designated by acapital letter (A, C, G, U); DNA is designated by a lower case letter(a, c, g, t); 2′OMe modification is designated by italics; lockednucleic acids (LNA) is designated by bold; 2′F is designated by asubscript “f”; phosphorothioate is designated by an underline; C6 aminois designated by a “N”; and 2′O-Propargyl G is designated by a “Y”.Table 1B gives the sequences of the complementary sequences (the targetsequences), Table 1C gives the formats and modifications for theinhibitors and Table 1D gives the corresponding sequences of theinhibitors.

The examples use the Homo sapiens (hsa) microRNA sequences hsa Mir21,hsa Mir let 7c, and hsa Mir 122 as the targets and various formats ofinhibitory molecules are produced to inactivate and/or inhibit thesemRNAs. The targets were chosen because they produce high levels ofmicroRNAs, so varying amounts of inhibition can be quantitated. Thegeneral sequences of the target microRNAs are as follows:

Mir-21: 5′-UAGCUUAUCAGACUGAUGUUGA-3′ (SEQ ID NO:144), Let-7a:5′-UGAGGUAGUAGGUUGUAUAGUU-3′ (SEQ ID NO:145), Let-7c:5′-UGAGGUAGUAGGUUGUAUGGUU-3′ (SEQ ID NO:146), and Mir-122:5′-UGGAGUGUGACAAUGGUGUUUG-3′ (SEQ ID NO:147). Note that Let-7C andLet-7a are from the same family and have almost identical sequences.They differ by just one nucleotide shown in bold and highlighted in thesequences. Table 1B shows the specific complementary sequences andmodifications used in the assays in the Examples.

TABLE 1B Complementary Sequences Sequence Complementary sequenceIdentifier UCAACAUCAGUCUGAUAAGCUA SEQ ID NO: 1 UCAACAUCAGUCUGAUAAGCUASEQ ID NO: 2 UCAACAUCAGUCUGAUAAGCUAc SEQ ID NO: 3 AACCAUACAACCUACUACCUCASEQ ID NO: 4 AACCAUACAACCUACUACCUCA SEQ ID NO: 5 AACCAUACAACCUACUACCUCAcSEQ ID NO: 6 AACUAUACAACCUACUACCUCAt SEQ ID NO: 7 AACUAUACAACCUACUACCUCASEQ ID NO: 8 AACCAUACAACCUACUACCUCAg SEQ ID NO: 9ACAAACACCAUUGUCACACUCCA c SEQ ID NO: 10 ACAAACACCAUUGUCACACUCCASEQ ID NO: 11 ACAAACACCAUUGUCACACUCCAc SEQ ID NO: 12AACCAUACAACCUACUACCUCA g SEQ ID NO: 13 AACUAUACAACCUACUACCUCAYSEQ ID NO: 14 AACUAUACAACCUACUACCUCA-Chol SEQ ID NO: 15UCAACAUCAGUCUGAUAAGCUAY SEQ ID NO: 16 UCAACAUCAGUCUGAUAAGCUA-CholSEQ ID NO: 17 AACUAUACAACCU A C T A C C T C At SEQ ID NO: 18 AA C U G UA C A AACUACUACCUCAt SEQ ID NO: 19 AAC_(f)U_(f) AU_(f) AC_(f)AAC_(f)C_(f)U_(f) AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) At SEQ ID NO: 20AAC_(f)U_(f) AU_(f) AC_(f) AAC_(f)C_(f)U_(f) AC_(f)U_(f)AC_(f)C_(f)U_(f)C_(f) At SEQ ID NO: 21

The miRNA inhibitors (see Tables 1C and 1D) were chemically synthesizedusing standard phosphoramidite-based nucleoside monomers and establishedsolid phase oligomerization cycles according to Beaucage, S. L. andIyer, R. P. (Tetrahedron, 1993 (49) 6123; Tetrahedron, 1992 (48) 2223).RNA phosphoramidites were protected with 2′O-TBDMS groups. Synthesis ofoligonucleotides was performed on a BioAutomation MerMade™ 192 orBioAutomation MerMade™ 12 synthesizer (BioAutomation Corp, Plano, Tex.).Eight equivalents of activator were used for every equivalent ofphosphoramidite to provide a satisfactory stepwise coupling yieldof >98% per base addition. Purification of the individualoligonucleotides for in vitro screening was carried out using highthroughput desalting and alcohol precipitation techniques. Purificationof the individual oligonucleotides for in vivo screening was performedwith either anion exchange or reverse-phase prep HPLC (Agilent 1200series) and oligonucleotides were desalted using a semi-permeablemembrane. Analytical HPLC (ion exchange or reverse-phase) was used fordetermining single strand purity, MALDI mass spectrometry was used fordetermining oligonucleotide identity, and UV spectroscopy was used forquantitative determination of inhibitors. When a cytidine (C) nucleosidewas modified as a bicyclo-sugar, the substituted nucleobase was a 5′methylated cytidine residue. When a uridine (U) nucleoside was modifiedas a bicyclo-sugar, the substituted nucleobase was a thymine (T)residue.

To test the mRNA inhibitors Biological Assay 1 and/or 2 were used (seeExamples 1 and 3). For Biological Assay 1, HeLa cells were pre-plated at10,000 per well in a 96-well plate. The next day 0.2 μl LIPOFECTAMINE®2000 transfection agent (Life Technologies) was complexed with 40 ng ofthe luciferase expression plasmid (with the corresponding miRNA bindingsite for let7c, miR21, or let7a; Life Technologies), 40 ng of the βGalexpression plasmid and 10, 20 or 100 nM of the microRNA inhibitors (ofdifferent designs). 24 hours later, the DUAL-LIGHT® assay was performedto determine how the microRNA inhibitors were able to up-regulateluciferase expression compared to other commercially available microRNAinhibitors. The βGal plasmid (Life Technologies) was used to normalizefor transfection efficiency.

The sequences of the microRNA inhibitors that were tested are shown inTable 1C, Table 1D and FIG. 10. The miRNA inhibitors were based on thefollowing target-binding sequences: Anti-mir-21 (synthetic inhibitor,reverse-complement to natural sequence):5′-UCAACAUCAGUCUGAUAAGCUA-3′(SEQ ID NO:148), Anti-let-7a (synthetic inhibitor, reverse-complement tonatural sequence):5′-AACUAUACAACCUACUACCUCA-3′ (SEQ ID NO:149),Anti-let-7c (synthetic inhibitor, reverse-complement to naturalsequence): 5′-AACCAUACAACCUACUACCUCA-3′ (SEQ ID NO:152), Anti-miR-122(synthetic inhibitor, reverse-complement to naturalsequence):5′-CAAACACCAUUGUCACACUCCA-3′(SEQ ID NO:150). The negativecontrol (neg) had the sequence: 5′-AAGUGGAUAUUGUUGCCAUCA-3′ (SEQ IDNO:151) and all of the nucleotides were 2′OMe. Inhibitor Y was purchasedfrom Dharmacon. Inhibitor X was purchased from Exiqon. See Tables 1A-1Dfor sequences and formats of the targets, the inhibitors, and thecontrols X, Y, 2′-OMe, and Neg.

Example 1 Initial Testing of Hairpin miRNA Inhibitors

This example provides novel hairpin miRNA inhibitor (anti-miR) formatsthat enabled strong inhibition of endogenous miRNAs. Inhibitors wereprepared for two different sequences: hsa mir21 and hsa mir let7c asdiscussed above. miRNA Inhibitors with non-nucleotide loops werechemically prepared using standard solid phase phosphoramidite chemistryprocedures and instrumentation for synthesis, cleavage, anddeprotection. The oligonucleotides were then purified utilizingprecipitation, desalting, and solid-phase extraction. Purifiedoligonucleotides were analyzed using analytical HPLC and massspectrometry.

The design of the inhibitors comprised the following (see FIG. 1): (i) atarget binding region (BR) with perfect sequence complementarity to theguide strand of a mature miRNA made up of 21-23 2′-O-methyl modifiednucleotides, and (ii) a flanking stem-loop structure at the 3′, 5′, orboth 3′ and 5′ termini composed of 4-8 nucleotide long stems (stem I andstem II) covalently attached 5′ to 3′ by a non-nucleotide loop (loop L1)such as polyethylene glycol (other potential loops: alkane diol,styrene, stilbene, triazole, tetrazole, peptide, polyamide, polyester,dithiol, polyamine, polyether, peptide nucleic acid, cycloalkane, polyalkene, or aryl). Target binding sequences of the Hairpin (HP)inhibitors were as follows:

Anti-miR-21: (SEQ ID NO: 148) 5′-UCAACAUCAGUCUGAUAAGCUA-3′ (22 nt),Anti-let-7c: (SEQ ID NO: 152) 5′-AACCAUACAACCUACUACCUCA-3′ (22 nt).

The Biological assay 1 is a reporter system for assaying anti-miRpotency using the pMIR-REPORT™ miRNA Expression Reporter Vector System(Life Technologies/Ambion, Austin, Tex.) (FIG. 2). This validatedreporter system contained two mammalian expression vectors:

(i) The pMIR-REPORT™ Luciferase miRNA Expression Reporter Vectorcontaining firefly luciferase (fLuc) under the control of a mammalianpromoter/terminator system and a target cloning region downstream of theluciferase translation sequence. This vector was used for cloning andtesting putative miRNA binding sites. It was also used to evaluateendogenous miRNA expression or the effects of either miRNA mimics (i.e.,Pre-miR™ miRNA Precursors) or miRNA inhibitors (i.e., Anti-miR™ miRNAInhibitors).

(ii) A second vector, pMIR-REPORT™ Beta-galactosidase Reporter ControlVector (Life Technologies) was used for normalization of transfectionefficiency.

FIG. 2 shows a schematic depiction of the Biological assay 1 used forevaluation of the performance of the miRNA inhibitors (anti-miRs). Theconcentrations of the miRNA inhibitors upon transfection were 30, 3, and0.3 nM. Initially, upon transfection of the pMIR-REPORT™ miRNAexpression reporter encoding firefly luciferase (fLuc) as well ascontaining the binding site for the particular miRNA, mRNA wastranscribed inside the cells. Endogenous miRNAs, naturally presentinside the cells, bound this mRNA and suppressed translation of thefirefly luciferase protein. As a result little or no fLuc was detectedin the sample. The same effect was observed if negative control anti-miR(non-targeting sequence) was co-transfected with the pMIR-REPORT™ miRNAexpression reporter. FIG. 2 shows the reporter expression in thepresence of the exogenous anti-miRs (miRNA inhibitors). In order toevaluate the efficacy of the miRNA inhibitors, the antisenseoligonucleotides were co-transfected with the pMIR-REPORT miRNAexpression reporter. (A pMIR-REPORT™ Beta-galactosidase Reporter ControlVector was also transfected in each well for normalization of deliveryefficiency.) mRNA was transcribed inside the cells, encoding fireflyluciferase as well as containing the binding site for the particularmiRNA. If the inhibitors bound and inactivated the endogenous miRNA(contained within Ago complex), no translational suppression of thefirefly luciferase protein occurred. As a result, high levels of fLucwere detected in the sample. By comparing the reading with the negativecontrol sample it was possible to calculate the average fold change foreach anti-miR format evaluated, and to determine which anti-miR causedthe maximum increase in the fLuc signal, i.e. was the most potent.

To test the hairpin (HP) anti-miRs, HeLa cells were pre-plated at 10,000per well in a 96-well plate in Dulbecco's Modified Eagle Medium (DMEM)high glucose (Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS)((MediaTech, Inc.) and 1% Penicillin (5000 Units)-Streptomycin (5000n)(Invitrogen). The next day, 0.2u1 LIPOFECTAMINE® 2000 transfection agentwas complexed with 40 ng of the pMIR-REPORT™ Luciferase miRNA ExpressionReporter Vector (with the corresponding miRNA binding site: miR21,let7c, let7a, or miR23), 40 ng of the pMIR-REPORT™ Beta-GalactosidaseReporter Control Vector and 0.003-100 nM of the chemically synthesizedanti-miR (of different designs). Twenty-four hours later, theDUAL-LIGHT® assay was performed to determine how the novel anti-miRswere able to up-regulate luciferase expression, compared to otheranti-miRs. The other anti-miRs used were a 2′-OMe modified antisenseoligonucleotide (2′-OMe), anti-miR Y (Dharmacon), and anti-miR X(Exiqon).

The Tropix® Dual-Light® Luminescent Reporter Gene Assay System forluciferase and β-galactosidase (Applied Biosystems, LLC, Foster City,Calif.) was used to quantitate firefly luciferase (fLuc) andβ-galactosidase (β-gal) activity in the same sample. First, theluciferase reporter enzyme activity was quantitated with an enhancedluciferase reaction. Following a 30-60 minute incubation and addition ofa light emission accelerator, β-galactosidase reporter enzyme activitywas quantitated with GALACTON-PLUS® substrate. The wide dynamic range ofthis dual assay enabled accurate measurement of fLuc and β-galactosidaseconcentrations over seven orders of magnitude (femtogram to nanogramrange).

By comparing the fLuc readings for specific anti-miR-transfected wellswith the negative control anti-miR (non-targeting sequence)-transfectedwells it was possible to calculate the average fold change for eachanti-miR format evaluated, and determine which anti-miR caused themaximum increase in the fLuc signal, i.e. was the most potent. Theaverage fold change was calculated in the following way: (fLuc activityanti-miR/βGal activity anti-miR)/(fLuc activity negative/βGal activitynegative).

The activity of the hairpin inhibitors HP#01-HP#09 (see Tables 1A-1D forformat and sequence of the inhibitors and targets) was evaluated usingthe pMIR-REPORT™ miRNA expression reporter (miR21 or let7c targetcloned), along with two controls: a 22 nt miRNA inhibitor with complete2′-O-methyl modification (2′-OMe), and miRNA inhibitor X (Exiqon) (FIG.3A and FIG. 3B). HP#01-HP#09 in FIG. 3A and FIG. 3B included hairpininhibitors with a 5′-loop, a 3′-loop or both 5′ & 3′-loops; PEG3(polyethyleneglycol polymer-3 monomeric units) or all-nucleotide loop;unmodified or 2′-OMe modified oligonucleotides (see also Tables 1A-1Cfor the sequences and formats).

The results of this experiment indicated the following: (1) Inhibitorswith single loops (i.e., loops on only one end) were effective forinhibiting miRNA function; (2) Inhibitors with 5′ loops were typicallymore potent than inhibitors with 3′-loop structures; (3) PEG loopsperformed better than the nucleotide loops for inhibitors with doubleloops and single loops; (4) 2′-OMe modified inhibitors were more potentthan unmodified 2′-OH inhibitors (5′-loop, 3′-loop and 3′& 5′ loops);and (5) LNA-modified antisense inhibitors worked better than2′OMe-modified antisense inhibitors of the same length 22 nt. (Note:more versions of stems and loops were tested in Examples 2, 3, 9 and10.)

Example 2 Effect of Linker Length, Linker Type and Stem Length on theActivity of the Hairpin Inhibitors

Additional hairpin inhibitors were prepared similarly as described inExample 1 and assayed using the same in vitro reporter system toinvestigate the effect of the linker length, linker type, and stemlength. As in Example 1, the efficiency of the hairpin inhibitorsHP#10-HP#31, HP#5, and HP#8 was evaluated using the pMIR-REPORT™ miRNAexpression reporter (miR21 or let7c target cloned), along with threecontrols: a 22 nt miRNA inhibitor with complete 2′-OMe modification(2′-OMe), and miRNA inhibitor X (Exiqon) and miRNA inhibitor Y(Dharmacon).

The formats of the HP#10-HP#31, HP#05, and HP#08 inhibitors are depictedin FIG. 1 with the following variations: a 5′-loop or a 3′-loop; 8versions of non-nucleotide loops and all-nucleotide loop; 3, 4, 5 bystem (see also Tables 1A-1D for formats and sequences).

The results are shown in FIG. 4A and FIG. 4B and indicated thefollowing: (1) Reverse complement oligonucleotides with 4-6 base-pairstems and non-nucleotide loops were potent inhibitors for in vitro lossof function studies of short (˜22 nts) regulatory non-coding RNAs,particularly microRNAs (miRNAs); (2) In general, hairpin inhibitors ofmultiple designs (different loops, stems) very efficiently inhibitedendogenous miRNAs and were more potent than the 2′-OMe modified miRNAinhibitor (2′-OMe), as well as miRNA inhibitor X (Exiqon) and miRNAinhibitor Y (Dharmacon); (3) Non-nucleotide loops were the same orbetter than all-nucleotide loops; L1-3, L7-8 (see Table 2) were best;(4) Inhibitors with 5′ loops were more potent than inhibitors with3′-loop structures; and (5) Inhibitors with longer stems (4-5 nt) weremore potent than those with 3 nt stems.

Example 3 Evaluation of the Potency of Hairpin Inhibitors

The potency of the hairpin inhibitors HP#10-HP#31, HP#5, and HP#8targeting let-7c miRNA was evaluated by quantification of the levels ofHMGA2 mRNA expression with TAQMAN® assays, along with three controls: a22 nt miRNA inhibitor with a complete 2′-OMe modification (2′-OMe), andmiRNA inhibitor X (Exiqon) and miRNA inhibitor Y (Dharmacon).

The formats of the HP#10-HP#31 inhibitors are depicted in FIG. 1 andTables 1A-1D. Variations of the hairpin inhibitors included: a 5′-loopor a 3′-loop; 8 versions of non-nucleotide loops and all-nucleotideloop; 3, 4, 5 by stem.

From the literature it was known that one of the direct endogenoustargets for miRNAs has-let7a and let7c, is the HMGA2 gene. A highlyefficient assay was developed for measuring the endogenous geneexpression levels to monitor potency of the miRNA inhibitors in vitro.Coupled with results from the reporter assays, this allowed for variousconclusions regarding inhibitor formats.

The Biological Assay 2, an endogenous assay, was used as follows: Totest the novel hairpin (HP) anti-miRs, HeLa cells were pre-plated at6,000 per well in a 96-well plate. The next day, 0.15u1 LIPOFECTAMINE®RNAIMAX™ transfection agent was complexed with 0.3-100 nM of thechemically synthesized anti-miR (of different designs) and added to thecells. 24 hrs later, cells were lysed with CELLS-TO-CT™ lysis buffer (10uL). HMGA2 mRNA levels were measured by qRT-PCR, using TAQMAN® GeneExpression CELLS-TO-CT™ kit (Applied Biosystems). A 20 μL RT reactionwas set up using 1 μL of the lysate (37° C. for 60 minutes, 95° C. for 5minutes, then 4° C.), followed by a 10 μL PCR reaction with a 2 μL cDNAinput using an inventoried TAQMAN® gene expression assay for HMGA2#.Samples were normalized with the Eukaryotic 18S rRNA endogenous control.

By comparing the values for anti-let7 transfected wells with thenegative control (non-targeting sequence) transfected wells it waspossible to calculate the relative HMGA2 expression (anti-let7/negative)for each anti-miR format evaluated. The higher the relative HMGA2expression—the more potent the miRNA inhibitor was.

The results (in FIG. 5) generally showed an increase of the HMGA2 mRNAlevels relative to the negative control-transfected samples. Morespecifically, the results of this experiment indicated the following:(1) In general, HP inhibitors of multiple designs (different loops,stems) very efficiently inhibited endogenous miRNAs and were more potentthan 2′-OMe modified miRNA inhibitor, as well as miRNA inhibitor X(Exiqon) and miRNA inhibitor Y (Dharmacon); (2) all non-nucleotide loopsperformed well (in particular L1 and L3), except L4, see Table 2 forloop structures; (3) inhibitors with 5′ loops were more potent thaninhibitors with 3′-loop structures; and (4) inhibitors with longer stems(>4 nt) were more potent than those with 3 nt stems.

Example 4 Evaluation of the Potency of Hairpin Inhibitor HP#24

The potency of the hairpin inhibitor HP#24 was evaluated byquantification of the fraction of the free endogenous miRNA (miR21 orlet7c) with miRNA-specific TAQMAN® assays, along with three controls: a22 nt miRNA inhibitor with complete 2′-OMe modification (2′-OMe), andmiRNA inhibitor X (Exiqon) and miRNA inhibitor Y (Dharmacon).

The format of the HP#24 inhibitor is depicted in FIG. 1 and Tables1A-1D. The results are shown in FIG. 6A-FIG. 6B.

One of the approaches to measure the efficiency of miRNA inhibition withantisense oligonucleotides was to measure the fraction of endogenousmiRNA remaining free (in the single-stranded form and thus accessiblefor primer binding) with TAQMAN® assays as follows:

Twenty four hours prior to transfection, HeLa cells were pre-plated at250,000 cells per well in 2.3 mL. The next day, synthetic miRNAinhibitors (targeting has-let7c, miR21, and a negative control) werediluted in OptiMEM® medium (Invitrogen, Carlsbad, Calif.) up to 100 μL.5 μL of LIPOFECTAMINE® 2000 transfection agent (Invitrogen, Carlsbad,Calif.) was diluted in OptiMEM® medium up to 100 μL and incubated atroom temperature for 5 minutes. Diluted transfection agent was mixedwith the inhibitors, incubated at room temperature for 20 minutes, thenthe 200 μL complex was added to the cells. The final volume in the wellswas 2.5 mL, and the miRNA inhibitor concentration was 3-100 nM.

At 24 hours post-transfection, cells were washed with 2 mL of PBS threetimes by rotating the plate to ensure coverage of the cells followed bytreatment with 400 μL 0.05% trypsin for 3 minutes at 37° C., then thetrypsin was inactivated by adding 1 mL of complete growth medium(DMEM+10% FBS). Media was swirled around and pipetted up and down todislodge cell clumps. The cells were transferred to 1.5 mL Eppendorftubes, pelleted at 800 g for 3 minutes, and the cell pellets were washedtwo times with 1 mL of PBS by flicking the tube to disrupt the pellet.The pellets were then lysed with 1.25 mL of the TAQMAN® MicroRNACELLS-TO-CT™ lysis buffer (Applied Biosystems). The lysates were mixedby inverting the tubes five times and then incubated at room temperaturefor 8 minutes. The lysis reactions were stopped by adding 125 μL StopSolution followed by inverting the tubes to mix and incubation at roomtemperature for another 2 minutes. Finally, RT and PCR were performed asdescribed for quantification of endogenous miRNA levels.

The assay for quantification of miRNAs consisted of two steps: reversetranscription (RT) and PCR. The RT primer from TAQMAN® MicroRNA Assaysfeatures a stem-loop design (Applied Biosystems Inc., Foster City,Calif.) (Chen et al, 2005, v.33, p. 1-9). A typical 10 μL RT reaction(Applied Biosystems) included either 10 nanogram of total purified RNAor 1 μL lysate prepared using the TAQMAN® MicroRNA Cells-to-CT™ kit and50 nM of RT primer. After adding the enzyme mix (at finalconcentrations, 0.25 mM of each dNTP, 3.33 units/μL of MULTISCRIBE™reverse transcriptase, 1×RT buffer, 0.25 units/μL of RNase inhibitor),the reaction mixture was incubated at 16° C. for 30 minutes, 42° C. for30 minutes, 85° C. for 5 minutes, and then 4° C. Real-time PCR wasperformed using a standard TAQMAN® PCR protocol on an Applied Biosystems7900HT Sequence Detection System. The 10 μL PCR reaction mixtureincluded 1 μL RT product, 1× TAQMAN® Universal PCR Master Mix, 0.2 μMTAQMAN® probe, 1.5 μM forward primer, and 0.7 μM reverse primer. Thereaction was incubated at 95° C. for 10 minutes, followed by 40 cyclesof 95° C. for 15 seconds and 60° C. for 1 minute.

The data in FIG. 6A-FIG. 6B are presented in the form of a bar graphsand showed a decrease of the free miRNA levels (available for primerhybridization and thus detection)-relative to negativecontrol-transfected samples for both targets. FIG. 6A shows the resultsfor let-7c and FIG. 6B shows the mir-21 results. The results of thisexperiment indicated that the hairpin inhibitor HP#24 was more potentthan the 2′-OMe modified miRNA inhibitor (2′-OMe), as well as miRNAinhibitor X (Exiqon) and miRNA inhibitor Y (Dharmacon).

This was the 3^(rd) biological assay that showed that HP inhibitorsperformed similarly or better than miRNA inhibitor X (Exiqon) and miRNAinhibitor Y (Dharmacon), and by far better than 2′-OMe antisenseinhibitors.

Example 5 Evaluation of the Potency of Hairpin Inhibitor HP#79-HP#83

The efficiency of the hairpin inhibitors HP#79-HP#83 was evaluated usingthe pMIR-REPORT™ miRNA expression reporter (miR21, let7a or let7c targetcloned), along with three controls: a 22 nt miRNA inhibitor withcomplete 2′-OMe modification (2′-OMe), and miRNA inhibitor X (Exiqon)and miRNA inhibitor Y (Dharmacon).

The formats of the HP inhibitors are depicted in FIG. 1 and Tables1A-1D. The variables were inhibitors with a 5′-loop or a 3′-loop; 0, 1,or 2 nt between the antisense region and the stem-loop structure.Biological evaluation using pMIR-REPORT™ miRNA Expression ReporterVector System was performed as described in Example 1.

The results shown in FIG. 7A-FIG. 7C indicated the following: (1) FormiRNA HP inhibitors with 5′-loops, introduction of an extra-nucleotidebetween the antisense region and the stem-loop structure did not reducethe inhibitor activity, i.e., the base stacking interaction with themiRNA strand was not crucial; (2) For HP inhibitors with 3′-loops,introduction of an extra-nucleotide between the antisense region and thestem-loop structure enhanced the anti-miR activity (compare withExample#1, where inhibitors with 3′-loops and 0 nt spacer were used);and (3) Overall, HP inhibitors of multiple designs very efficientlyinhibited miRNAs. Depending on the miRNA target and assay used(reporter, endogenous, etc) the best format varied.

Advantages of the Hairpin Inhibitors:

The hairpin inhibitors described herein utilizing an asymmetric designfor the inhibitor, with a single stem loop at the 3′ or 5′ end of theantisense region and/or wherein the stem loop had a non-nucleotide loopwere compared. The asymmetric design had more potency then the twocommercially-available inhibitors used to compare. The hairpininhibitors were 34-36 nucleotides long making the manufacturing of suchmolecules more feasible at a lower cost, especially if using anon-nucleotide loop. Contrary to current ideas, this suggests that thebase-stacking interaction is not as crucial as was thought. Withoutbeing bound by a specific explanation for the increased potency of theasymmetric hairpin inhibitors, the process was likely driven primarilynot by hybridization of the miRNA-antisense, but by the Ago protein(which contains an miRNA guide strand) which has certain templatepreferences (size, structure, etc). The natural targets of the Agoprotein are certain mRNAs.

The addition of a 3′ alkyl amino group somewhat enhanced potency atlower concentrations. Comparison of inhibitors with non-nucleotide loopsat the 3′ end prepared with and without a 3′ alkyl amino group showedthat inhibitors with alkyl amino modification demonstrated betteractivity at very low concentrations (0.03 nM).

Example 6 Evaluation of the Potency of Modified Antisense Inhibitors

The efficiency of the modified antisense oligonucleotide-basedinhibitors BTM#03-BTM#22 was evaluated using pMIR-REPORT™ miRNAexpression reporter (miR21, let7a or let7c target cloned), along withthree controls: a 22 nt miRNA inhibitor with complete 2′-OMemodification (2′-OMe), and miRNA inhibitor X (Exiqon).

The formats of the antisense inhibitors are depicted in FIG. 1 andTables 1A-1D. The variables were inhibitors with combinations of LNA,2′-F, 2′-OMe modification; 21 nt versus 15 nt long antisense sequence.Synthesis of miRNA inhibitors and biological evaluation usingpMIR-REPORT™ miRNA Expression Reporter Vector System was performed asdescribed in Example 1. The results are shown in FIG. 8A-FIG. 8C.

miRNA inhibitors were prepared that were highly chemically modified(LNA, ENA and 2′-F combinations with 2′-OMe) without any hairpinstructures- to compare the value of strong binding chemistrynucleotides. The results showed that: (1) LNA chemistry and LNA/2′-OMe,LNA/2′-F combinations were more effective at inhibition of miRNAs than2′-OMe or 2′-F anti-miR modifications; (2) inhibitors with thefull-length antisense sequence (21 nt) were significantly more potentthan inhibitors with the shorter antisense region (15 nt); (3) modified22 nt miRNA inhibitors were either as good or worse than the hairpininhibitors in Examples 1 and 2); and (4) LNA modifications incorporatedinto the hairpin inhibitors may improve their potency. However, LNAmodified molecules are challenging to synthesize and can be expensive.

Example 7 Evaluation of the Potency of Multimerized Inhibitors

The design of the miRNA inhibitor multimers targeting miR21 is depictedin FIG. 9 and FIG. 10. Biological evaluation using pMIR-REPORT™ miRNAExpression Reporter Vector System was performed as described inExample 1. The results are shown in FIG. 11.

Multimerized anti-miRs were designed and tested to determine theirperformance. As shown in FIG. 10, 30-45 nucleotide long anti-miRs weresynthesized (enhanced with chemical modifications for stronger binding)so that the central 20-22 nt part was complementary to the target miRNAstrand, and flanking sequences were used for multimeric complexformation. Upon addition of the “bridging” oligonucleotide (NBR2c:NBR1cin FIG. 9) (with or without LNA or other modifications to promotestronger interaction) the anti-miRs spontaneously formed multimericcomplexes.

Multimeric Anti-miRs targeting mir21, prepared under differentconditions, were co-transfected into HeLa cells at a concentration of 10nM with the luciferase expression plasmid containing the mir21 bindingsite and βGal plasmid (used for normalization of transfectionefficiency). 24 hours later the DUAL-LIGHT® Assay was performed tomonitor the effect of the anti-miR on luciferase expression. Eachanti-miR was normalized to the negative control anti-miR(NCα).Multimeric complex formation was as follows: Antisense oligonucleotide(with extra-sequences at the 3′-termini and 5′-termini—enabling complexformation, see FIG. 9 and FIG. 10) was annealed with the “bridging”oligonucleotide at 95° C. for 3 min, followed by a 1 hour incubation at37° C. The designations #1, #2, and #3 of FIG. 11 refer to differentannealing buffers. Buffer #1: 10 mM Tris-HCl pH 7.4, 100 mM NaCl. Buffer#2: 6 mM Hepes pH 7.4, 20 mM potassium acetate, 0.4 mM Magnesiumacetate. Buffer#3: 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1 mM EDTA. Thedesignations 1×, 2×, and 4× refer to the molar excess of the “bridging”oligonucleotide over the long antisense oligonucleotide. The multimericmiRNA inhibitors self assembled in the specified buffer. The number ofmultimers was dependent upon the type of buffer used and the molarexcess of the bridging oligonucleotide.

These anti-miRs had several attractive features: (1) The large sizeclosely mimicked the natural substrate-mRNA- and thus promoted veryefficient binding with miRNA/Ago; (2)

Multimers did not have secondary structure that could complicate complexformation with miRNAs-in contrast to “miRNA sponges” (long tandemantisense transcripts expressed from DNA vectors); (3) The termini ofmultimers had extra protection from RNases; (4) Packaging and in vitrodelivery of the multimers was more efficient when using certaincommercially available reagents (such as PEI=polyethyleneimine), becausecomplexes are long and rigid similarly to dsDNA; and (5) The multimerswere chemically synthesized so modifications could be incorporated,enabling strong interaction with the miRNA target (2′-OMe was used, butLNA or other molecules could be used)—in contrast to “miRNA sponges”.

A long anti-miR to mir21(45 nt)-the core 22-nt RNA, 2′OMe modified,flanked by lint and 12 nt sequences, was also synthesized. The“bridging” oligonucleotide was 21 nt long, LNA was modified at everyother position. Multimeric complexes were formed by mixing equal amountsof the long anti-miR and “bridging” oligonucleotide in annealing buffer(10 mM Tris pH 7.4, 100 mM NaCl), incubating at 95° C. for 3 minutes andcooling down at 37° C. for 1 hr. The biological evaluation usingpMIR-REPORT™ miRNA Expression Reporter Vector System was performed asdescribed in Example 1.

Initial evaluation of the performance of the multimers showed that theyhad better inhibitory activity than 2′-OMe anti-miRs, as well as miRNAinhibitor X (Exiqon) and miRNA inhibitor Y (Dharmacon) (FIG. 11).

Example 8 In Vivo Studies

Hairpin inhibitor miR122 was tested in vivo as follows: Mice (3animals/group) were injected in the tail vein with 50 mg/kg anti-miR122(with phosphorothioate modifications, without any delivery reagent), on3 subsequent days, and sacrificed 24 h post 3rd injection. Levels ofAldoA, Hfe2, Slc35a4, Lass6 mRNAs (reported targets for miR-122: Elmenet al, 2008 Nucl. Acids Res. v.36, p. 1153-1162; Davis et al, Nucl.Acids Res. 2009 v.37, p. 70-77) in their livers was quantified byqRT-PCR. Data normalized to untreated mice, and Let7-additional “negcontrol”—is shown. MiR122 5′HPp=s was the format HP#81, with completephosphorothioate (P═S) modification of the target binding strand for invivo use. MiR122 3′HPp=s was the format HP#83, with completephosphorothioate (P═S) modification of the target binding strand for invivo use (MiR122 5′HP format HP#81 w/o phosphorothioate modification didnot show any activity).

The results in FIG. 12 showed the following: (1) miRNA inhibitors withphosphorothioate modifications could be used in in vivo experiments, andthey induced robust inhibition of their targets in the liver, uponsystemic tail vein administration. (2) phosphorothioate modificationswere advantageous for in vivo experiments. Formats that were notself-deliverable in vivo could be successfully used with in vivodelivery reagents. (3) miRNA inhibitors with 5′-hairpin structures hadbetter activity than inhibitors with 3′-hairpins in vitro and in vivo.

Example 9 Evaluation of the Potency of Hairpin Inhibitors

The potency of hairpin inhibitors HP#79, HP#81, HP#96, HP#85, HP#88,HP#93, and HP#96 targeting let-7a miRNA with CLICK-IT® loops (HP#85) orCLICK-IT® chemistry at the 3′-termini (HP#88; ready for conjugation ofother molecules, e.g., cholesterol or dye label) were tested. See Tables1A-1D for the format and sequence of the inhibitors and/or the targets.Click chemistry is a class of high yielding reactions for generating newcompounds. The most popular reaction from the class is the coppercatalyzed 1,3 dipolar cycloaddition between an alkyne and an azide toyield a substituted 1,2,3, triazole (also known as Huisgencycloaddition). Potency was evaluated by quantification of the levels ofHMGA2 mRNA expression with TAQMAN® assays, along with three controls: a22 nt miRNA inhibitor with complete 2′-OMe anti-miRs, as well as miRNAinhibitor X (Exiqon) and miRNA inhibitor Y (Dharmacon) as discussed inExamples 1 and 4. The concentration of miRNA inhibitors upontransfection was 0.3, 3, 30 nM. Increase of the HMGA2 mRNA levels isshown relative to negative control-transfected samples. The increase inthe firefly luciferase expression induced by miRNA inhibition (theAverage fold change) was calculated in the following way: (fLuc activityanti-miR/βGal activity anti-miR)/(fLuc activity neg/βGal activity neg).(For details, see Example 1 Materials and Methods section.)

The results in FIG. 13 and FIG. 14 showed that (1)-hairpin inhibitorssynthesized with CLICK-IT® loops (HP#85) or CLICK-IT® chemistry at the3′-termini (HP#88; ready for conjugation of other molecules, e.g.cholesterol or dye label) have the same potency as the HP inhibitors(2)-HP conjugates with cholesterol (HP#96) are functional, and the levelof activity is similar to unconjugated HP. Cholesterol can enable invitro and in vivo delivery of these inhibitors.

Example 10 Evaluation of the Potency of Hairpin Inhibitors

HP#89, HP#90, HP#91, HP#92: LNA and 2′F/2OMe modified hairpin inhibitorstargeting let-7c miRNA were tested for potency by quantification of thelevels of HMGA2 mRNA expression with TAQMAN® assays, along with threecontrols: a 2′-OMe anti-miR, as well as miRNA inhibitor X (Exiqon) andmiRNA inhibitor Y (Dharmacon). See Tables 1A-1D for formats andsequences of the inhibitors and targets. The assays were performed as inExample 4. The concentration of the miRNA inhibitors tested upontransfection was 10 nM.

The results are shown in FIG. 15 where an increase of the HMGA2 mRNAlevels is shown relative to the negative control-transfected samples.The results showed that (1) chemistries other than 2′-OMe can be usedfor HP inhibitors, and all of them are highly functional. However, LNAor other modifications did not result in superior performance ofinhibitors, compared to 2′-OMe modifications. However, the activity ofthe inhibitors was at such a high level already, improvements may bevery small or negligible. With hairpin structures 2′-OMe modificationsperform very well, but for single-stranded antisense oligonucleotidesthe LNA-modified inhibitors were superior to 2′-OMe inhibitors. Thus,the modifications that enhanced activity varied for differentstructures. However, in all cases, the hairpin inhibitors weresignificantly better than “standard” 2′-OMe inhibitors (21 nt long) andX and Y inhibitors (Exiqon and Dharmacon).

Example 11 Synthesis of inhibitor with Non-nucleotide Loop L11 thatTargets Let 7a miRNA

Inhibitor HPX (SEQ ID NO:103) was synthesized with non-nucleotide loopL11 (see Table 2) via copper catalyzed Huisgen 1,3-dipolarcycloaddition. The miRNA inhibitor targeted Let 7a miRNA. However thismethod can be used to synthesize any type of inhibitor. The sequence andchemical structure of compound 1 is shown below. Synthesis was achievedusing solid phase phosphoramidite synthesis on a MerMade™ 192synthesizer. Standard coupling times and cycles were employed along withI₂/H₂O oxidation and acetic anhydride capping on DNA T nucleoside loadedCPG columns 1 mmole (Biosearch). 2′OMe phosphoramidites A, C, G, and Uwere purchased from Chemgenes, Inc and diluted with anhydrousacetonitrile prior to use. The phosphoro-hexyne moiety was installedusing 5′ alkyne modifier (Glen Research). After synthesis, the CPG wastreated with a mixture of aqueous ammonia and methylamine for 15 minutesto cleave the oligonucleotide from the solid support. This mixture wasfurther incubated for a period of 2.5 hours in the ammonia/methylaminesolution to afford the removal of both the exocyclic amino protectiongroups (benzyl for A, acetyl for C, and dmf for G) as well as thecyanoethyl protecting groups from the phosphate backbone. The aqueoussolution was dried via compressed air under elevated temperature toproduce the crude product. The oligonucleotide was purified on anionexchange resin using an Agilent 1200 HPLC with NaClO₄ buffer as theeluent. The oligonucleotide was then desalted utilizingultracentrifugation cartridges (Sartorius, 2000 MWCO) and the molecularweight of the oligonucleotide was verified with MALDI (AppliedBiosystems).

The sequence and chemical structure of compound 2 is shown below.Synthesis was achieved using solid phase phosphoramidite synthesis on aMerMade™ 192 synthesizer. Standard coupling times and cycles wereemployed along with I₂/H₂O oxidation and acetic anhydride capping on C6phthalimido loaded CPG (Prime Synthesis) 500 nmole columns. 2′OMephosphoramidites A, C, G, and U were purchased from Chemgenes, Inc anddiluted with anhydrous acetonitrile prior to use. After synthesis, theCPG was treated with a mixture of aqueous ammonia and methylamine for 15minutes to cleave the oligonucleotide from the solid support. Thismixture was further incubated for a period of 2.5 hours in theammonia/methylamine solution to afford the removal of both the exocyclicamino protection groups (benzyl for A, acetyl for C, and dmf for G) aswell as the cyanoethyl protecting groups from the phosphate backbone.The aqueous solution was dried via compressed air under elevatedtemperature to produce the crude product. The oligonucleotide waspurified on anion exchange resin using an Agilent 1200 HPLC with NaClO₄buffer as the eluent. The oligonucleotide was then desalted utilizingultracentrifugation cartridges (Sartorius, 2000 MWCO) and molecularweight of the oligonucleotide was verified with MALDI (AppliedBiosystems).

The sequence and chemical structure of compound 3 is shown below.Azidobutyrate NHS ester (Glen Research) in dimethylsulfoxide (15 μL,0.17 M) was added to 0.5 umole of compound 2 in 100 mM sodium boratebuffer. The solution was mixed using an orbital shaker at roomtemperature for a period of 4 hours. The oligonucleotide was purified onanion exchange resin using an Agilent 1200 HPLC with NaClO₄ buffer asthe eluent. The oligonucleotide was then desalted utilizingultracentrifugation cartridges (Sartorius, 2000 MWCO).

The sequence and chemical structure of miRNA Inhibitor SEQ ID NO:103 isshown below. CuSO₄ (10 μL, 0.1 M), butanone (20 μL), andTri[(1-benzyl-1,2,3-triazol-4-yl)methyl]amine (TBTA, 5 μL, 0.1 M) wasadded to a solution of 1 (0.05 μmol, 0.7 mM) in phosphate bufferedsaline. After vortexing for 1 minute, compound 3 (84 μL, 0.9 M) andascorbic acid (2 μL, 0.5 M) were added to the mixture and the solutionwas vortexed again for 1 minute. The solution was then mixed using anorbital shaker at room temperature for a period of 2 hours. Theinhibitor was then desalted utilizing ultracentrifugation cartridges(Sartorius, 2000 MWCO) and molecular weight of the inhibitor wasverified with MALDI (Applied Biosystems).

Example 12 Synthesis of inhibitor with Non-Nucleotide Loop L11 thatTargets mir21 miRNA

Inhibitor HPX (SEQ ID NO:106) was synthesized with non-nucleotide loopL11 (see Table 2) via copper catalyzed Huisgen 1,3-Dipolar cycloadditionof the non-nucleotide loop. This inhibitor targets mir21 miRNA.

The sequence and chemical structure of compound 4 is shown below.Synthesis was achieved using solid phase phosphoramidite synthesis on aMerMade 192 synthesizer. Standard coupling times and cycles wereemployed along with I₂/H₂O oxidation and acetic anhydride capping on DNAC nucleoside loaded CPG columns 1 μmole (Biosearch). 2′OMephosphoramidites A, C, G, and U were purchased from Chemgenes, Inc anddiluted with anhydrous acetonitrile prior to use. The phosphoro-hexynemoiety was installed using 5′ Alkyne modifier (Glen Research). Aftersynthesis, the CPG was treated with a mixture of aqueous ammonia andmethylamine for 15 minutes to cleave the oligonucleotide from the solidsupport. This mixture was further incubated for a period of 2.5 hours inthe ammonia/methylamine solution to afford the removal of both theexocyclic amino protection groups (benzyl for A, acetyl for C, and dmffor G) as well as the cyanoethyl protecting groups from the phosphatebackbone. The aqueous solution was dried via compressed air underelevated temperature to produce the crude product. The oligo waspurified on anion exchange resin using an Agilent 1200 HPLC with NaClO₄buffer as the eluent. The oligonucleotide was then desalted utilizingultracentrifugation cartridges (Sartorius, 2000 MWCO) and molecularweight of the oligonucleotide was verified with MALDI (AppliedBiosystems).

miRNA Inhibitor SEQ ID NO:154. CuSO₄ (10 μL, 0.1 M), butanone (20 μL),and Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 5 μL, 0.1M) was added to a solution of 4 (0.05 mmol, 0.56 mM) in phosphatebuffered saline. After vortexing for 1 minute, compound 3 from ExampleXI (84 μL, 0.9 M) and ascorbic acid (2 μL, 0.5 M) were added to themixture and the solution was vortexed again for 1 minute. The solutionwas then mixed using an orbital shaker at room temperature for a periodof 2 hours. The inhibitor was then desalted utilizingultracentrifugation cartridges (Sartorius, 2000 MWCO) and molecularweight of the inhibitor was verified with MALDI (Applied Biosystems).

Example 13 Effect of Loop Length on the Activity of the HairpinInhibitors

Additional hairpin inhibitors with PEG5 loops and 6 bp stems wereprepared similarly to those described in Example 1 and assayed using thesame in vitro reporter system to investigate the effect of the looplength on the inhibitor potency. The efficiency of the hairpininhibitors HP#130 (PEG5) was evaluated side by side with HP#81 (PEG6),using the pMIR-REPORT™ miRNA expression reporter (miR21 or let7a targetcloned), along with a positive control-a 22 nt miRNA inhibitor withcomplete 2′-OMe modification (2′-OMe), and a negative control. Theformats of the HP#130 and HP#81 inhibitors are depicted in FIG. 1. Notethat the only difference was the length of their non-nucleotide loop.Thus, the sequence of HP#130 is the same as that of HP#81. In FIG. 16,the expression induced by miRNA inhibition was the Average fold changeand was calculated in the following way: (fLuc activity anti-miR/βGalactivity anti-miR)/(fLuc activity negative/βGal activity negative).Note: the higher the bars—the stronger miRNA inhibition. Theconcentration of the miRNA inhibitors upon transfection was 0.3 and 3 nMand all experiments were performed in triplicate.

The results in FIG. 16 indicated that microRNA inhibitors with PEG6 andPEG5 loops displayed about the same in vitro activity for miR21 andlet7a targets, and both were superior to the standard antisenseinhibitor without the stem-loop structure.

TABLE 3 Sequences in FIG.S 16-20 SEQ loop ID posi- Linker LinkerComplementary FIG. NO. tion HR I (L1) HR II HR III (L2) HR IV Targetsequence 16 089 HP81 5′ GGCAC L3 CGUGC na na na mir 21 SEQ ID NO: 003 GC 16 139 HP130 5′ GGCAC L12 CGUGC na na na mir 21 SEQ ID NO: 003 G C 16031 2′OMe none na na na na na na mir 21 UCAACAUCAGUCUGAUAAGCUAN(SEQ ID NO: 31) 16 064 Neg neg AAGUGGAUAUUGUUGCCAUCAN (SEQ ID NO: 64) 16134 HP81 5′ GGCAC L3 CGUGC na na na let7a SEQ ID NO: 007 G C 16 140HP130 5′ GGCAC L12 CGUGC na na na let7a SEQ ID NO: 007 G C 16 097 2′OMenone na na na na na na let7a AACUAUACAACCUACUACCUCAN (SEQ ID NO: 97) 16064 Neg neg AAGUGGAUAUUGUUGCCAUCAN (SEQ ID NO: 64) 17 141 HP130 5′ GGCACL12 CGUGC na na na mir122 SEQ ID NO: 012 G C 17 064 Neg negAAGUGGAUAUUGUUGCCAUCAN (SEQ ID NO: 64) 18 umod 18 031 2′OMe none na nana na na na mir 21 UCAACAUCAGUCUGAUAAGCUAN (SEQ ID NO: 31) 18 089 HP815′ GGCAC L3 CGUGC na na na mir 21 SEQ ID NO: 003 G C 18 091 HP83 3′ nana na UCCGU L3 GCAC mir 21 SEQ ID NO: 001 GC Gg 18 Y mir 21 18 umod 18041 2′OMe none na na na na na na let7c AACCAUACAACCUACUACCUCAN(SEQ ID NO: 41) 18 100 HP81 5′ GGCAC L3 CGUGC na na na let7cSEQ ID NO: 006 G C 18 102 HP83 3′ na na na UCCGU L3 GCAC let7cSEQ ID NO: 004 GC Gg 18 Y let7c 19 089 HP81 5′ GGCAC L3 CGUGC na na namir 21 SEQ ID NO: 003 G C 19 064 Neg neg AAGUGGAUAUUGUUGCCAUCAN(SEQ ID NO: 64) 19 134 HP81 5′ GGCAC L3 CGUGC na na na let7aSEQ ID NO: 007 G C 19 064 Neg neg AAGUGGAUAUUGUUGCCAUCAN (SEQ ID NO: 64)20 142 HP81 5′ GGCAC L3 CGUGC na na na mir23a GGAAAUCCCUGGCAAUGUGAUc G C(SEQ ID NO: 156) 20 X 20 064 Neg neg AAGUGGAUAUUGUUGCCAUCAN(SEQ ID NO: 64) 20 089 HP81 5′ GGCAC L3 CGUGC na na na mir 21SEQ ID NO: 003 G C 20 X 20 064 Neg neg AAGUGGAUAUUGUUGCCAUCAN(SEQ ID NO: 64) 21 143 2′Ome mir122 ACAAACACCAUUGUCACACUCCAN(SEQ ID NO: 157) 21 144 HP81 5′ GGCAC L3 CGUGC na na na mir122SEQ ID NO: 012 21 145 An- mir122 ACAAACACCAUUGUCACACUCCA- tago Chol mir(SEQ ID NO: 162) 21 064 Neg neg AAGUGGAUAUUGUUGCCAUCAN (SEQ ID NO: 64)23 146 HP115 5′ GGCAC L3 CGUGC na na na mir122 ACAAACACCAUUGUCACACUCCA-Chol (SEQ ID NO: 162)

Example 14 Evaluation of the Potency of HP#130 (PEG5) Inhibitor

The potency of the miR122-targeting HP#130 inhibitor with the PEG5 loopwas evaluated in exogenous assays. The assays were similar to thosedescribed in Example 1. The only difference was that for miR 122, fourconstructs were used with cloned natural targets for miR122-RIMS1,GNPDA2, ANKRD13C, and G6PC3 fragments; in the above-described Examples#1-13, all in vitro reporters contained the cloned miRNA binding sitefully complementary to the miRNA of interest. The HP#130 inhibitors wereco-transfected with these constructs, and 24 h later the Luciferasesignal was measured versus a negative control-enabling the determinationof the potency of the inhibitors towards these four targets. The resultsare shown in FIG. 17. In the figure, the expression induced by miRNAinhibition was the Average Fold change and was calculated in thefollowing way: (fLuc activity anti-miR/βGal activity anti-miR)/(fLucactivity negative/βGal activity negative). The concentration of miRNAinhibitors upon transfection was 3 nM. All experiments were performed intriplicate. The results in FIG. 17 indicated that the miR122 inhibitorwas active on all four natural miR122 targets, displaying 1.5-3 foldupregulation.

Example 15 Detection of the HP#81 Hairpin Inhibitor with Small RNATAQMAN® Assays

For in vitro and in vivo experiments, understanding the efficiency ofmiRNA inhibitor intracellular delivery, localization and distribution,helps to unravel the pathways and kinetics of the process. Small RNATAQMAN® assays (Applied Biosystems/Life Technologies) enable accurateand robust quantification of small RNA molecules, including miRNAinhibitors. The purpose of this example was to explore whether chemicalmodifications and the 5′-hairpin structure would negatively impact thedetection of these molecules. HP#81 inhibitors (with 5′-stem/loop andcomplete 2′-OMe modifications) were compared to the unmodified antisenseoligonucleotides, antisense oligonucleotides with complete 2′-OMemodification, inhibitors with 3′-stem/loop and complete 2′-OMemodifications, and miRNA inhibitor Y (Dharmacon) featuring both 3′- and5′-terminal loops and complete 2′-OMe modifications. The miRNAinhibitors in the above listed formats were synthesized for miR21 andlet7c, and detected with TAQMAN® assays in a cell-free system.

The assay for quantification of miRNA inhibitors consisted of two steps:reverse transcription (RT) and PCR. The RT primer from TAQMAN® MicroRNAAssays features a stem-loop design (Applied Biosystems Inc., FosterCity, Calif.). A typical 10 μL RT reaction included 0.5 pmol of miRNAinhibitor and 50 nM of RT primer. The anti-miR and stem loop primermixture was heat denatured, incubated at 85° C. for 5 min, 60° C. for 5min, and transferred to ice. After adding the enzyme mix (at finalconcentrations, 0.25 mM of each dNTP, 3.33 units/μL of MultiScribe™reverse transcriptase, 1×RT buffer, 0.25 units/μL of RNase inhibitor),the reaction mixture was incubated at 16° C. for 30 minutes, 42° C. for30 minutes, 85° C. for 5 minutes, and then 4° C. Real-time PCR wasperformed using a standard TAQMAN® PCR protocol on an Applied Biosystems7900HT Sequence Detection System. The 10 μL, PCR reaction mixtureincluded 1 μL, RT product, 1× TAQMAN® Universal PCR Master Mix, 0.2 μMTAQMAN® probe, 1.5 μM forward primer, and 0.7 μM reverse primer. Thereaction was incubated at 95° C. for 10 minutes, followed by 40 cyclesof 95° C. for 15 seconds and 60° C. for 1 minute.

The results are shown in FIG. 18. As expected, the unmodified antisenseoligonucleotides were detected most efficiently (as indicated by lowestCt values). Antisense oligonucleotides with complete 2′-OMe modificationwere detected slightly less efficiently. HP#81 inhibitors (with5′-stem/loop and complete 2′-OMe modifications) were also detectedslightly less efficiently, with a Ct difference of <2 versus unmodifiedsingle-stranded RNA. Inhibitors with 3′-stem/loop and complete 2′-OMemodifications, and especially the miRNA inhibitor Y (Dharmacon)featuring both 3′- and 5′-terminal loops and complete 2′-OMemodifications were detected much less efficiently, presumably becausethe loop structure at the 3′-end interfered with RT primer binding andelongation. To summarize, HP#81 miRNA inhibitors were compatible withstem-loop RT primers and Small RNA TAQMAN® assays (LT) and thus could beeasily traced and quantified in the in vitro and in vivo experiments.

Example 16 Specificity of the Hairpin Inhibitors

The specificity of the HP#81 inhibitors (with PEG6 loops and 6 bp stems)was studied. An inhibitor (HP#81 format, SEQ ID NO:89) for miR-21 wastested against its intended target, miR-21, as well as miR-31, let-7a,miR-106a, miR-23a, miR-19a, miR-17, and miR-24 targets. An inhibitor(HP#81 format, SEQ ID NO:134) for let-7a was tested against its intendedtarget, let-7a, as well as miR-31, miR-21, miR-106a, miR-23a, miR-19a,miR-17, and miR-24 targets. The experimental conditions for theseexogenous assays were similar to those described in Example 1.

In FIG. 19A and FIG. 19B, the expression induced by miRNA inhibition wasmeasured as the Average Fold change and was calculated in the followingway: (fLuc activity anti-miR/βGal activity anti-miR)/(fLuc activitynegative/βGal activity negative). The concentration of miRNA inhibitorsupon transfection was 3 nM. All experiments were performed intriplicate. The results shown in FIG. 19A and FIG. 19B indicated thefollowing: HP#81 microRNA inhibitors were specific to their intendedtarget and did not affect the levels of the unrelated miRNA targets.

Example 17 Stability of the Hairpin Inhibitors

Long-term stability of the HP#81 inhibitors (with PEG6 loops and 6 bpstems) was evaluated. Water solutions (20 μmolar) of the inhibitorstargeting miR-21 (SEQ ID NO:89) and miR-23a (SEQ ID NO:142) were storedat +4° C. for 1 week, 2 months and 1 year. Their potency was thenevaluated side-by-side with miRNA inhibitor X (Exiqon) and negativecontrol. The experimental conditions for these exogenous assays weresimilar to described in Example 1: miRNA inhibitors were co-transfectedin HeLa cells with reporter constructs, and the Luciferase readoutallowed determination of their potency towards endogenous miRNAs.

The expression induced by miRNA inhibition was the Average fold changeand was calculated in the following way: (fLuc activity anti-miR/βGalactivity anti-miR)/(fLuc activity negative/βGal activity negative).Note: the higher the bars—the stronger the miRNA inhibition. Theconcentration of miRNA inhibitors upon transfection: 0.3 and 3 nM. Allexperiments were performed in triplicate.

The results in FIG. 20A-FIG. 20B indicated that the HP#81 microRNAinhibitors were stable for prolonged periods of time, and there was noloss of potency after 1 year real-time storage of stock solutions inwater, unfrozen, at +4° C.

Example 18 In Vivo Performance of the Hairpin Inhibitors

Hairpin inhibitor MiR122 was tested in vivo as follows: Mice (3animals/group) were injected in the tail vein with 5 mg/kg HP#81anti-miR122 (without p=s modifications) complexed with INVIVOFECTAMINE®2.0 reagent (Life Technologies) according to the manufacturer'sprotocol, on 3 subsequent days, and sacrificed 24 h post 3rd injection.

The 2nd group of mice was similarly treated with single-stranded 2′-OMemiRNA inhibitors complexed with INVIVOFECTAMINE® 2.0 reagent.

The 3rd group of mice was injected with miR122 antagomirs (Krutzfeldt J.et al., Nucl. Acids Res. 2007 v.35, p. 2885-2892) at 50 mg/kg, giventhree daily injections. MiR122 antagomirs were self-deliverablechemically modified siRNAs conjugated to cholesterol, and they wereinjected without any delivery reagent.

The 4th group of mice was a negative control and the mice were injectedwith negative control (non-targeting) miRNA inhibitor, complexed withINVIVOFECTAMINE° 2.0 reagent.

The 5th group of mice was the untreated group (normal uninjectedanimals). All experiments were performed with 3 animals per group. Afterthe animals were sacrificed, levels of AldoA, Hfe2, S1c35a4, Lass6 mRNAs(reported targets for miR-122: see Elmén et al, 2008 Nucl. Acids Res.v.36, p. 1153-1162; and Davis et al, Nucl. Acids Res. 2009 v.37, p.70-77) in the livers were quantified by qRT-PCR. mRNA upregulation wasnormalized to the negative control oligo-injected mice (=100%).

The results in FIG. 21 showed the following: (1) miRNA inhibitors wereefficiently delivered to the liver upon systemic tail veinadministration with INVIVOFECTAMINE® 2.0 reagent and inhibited microRNA122 as indicated by upregulation of four targets. (2) HP#81 inhibitorswere superior to 2′-OMe antisense oligonucleotides. (3) HP#81 inhibitorsdelivered with INVIVOFECTAMINE® 2.0 reagent induced better miRNAinhibition compared to Antagomirs-despite the fact that antagomirconjugates were used at a 10-fold higher dose.

Example 19 In Vivo Performance of the Cholesterol-Modified HairpinInhibitors

Performance of the sterol-modified hairpin inhibitors for miR122 wasevaluated in vivo. The format of miRNA inhibitor HP#115 (SEQ ID NO:146)is based on HP#81 but contains phosphorothioate modifications and isconjugated to cholesterol. Mice (3 animals/group) were injected in thetail vein with 50 mg/kg anti-miR122 HP#115 (without any deliveryreagent), on 3 subsequent days, and sacrificed 24 h post 3rd injection.Levels of AldoA, Hfe2, S1c35a4, Lass6 mRNAs (four reported targets formiR-122: Elmén et al, 2008 Nucl. Acids Res. v.36, p. 1153-1162; Davis etal, Nucl. Acids Res. 2009 v.37, p. 70-77) in their livers werequantified by qRT-PCR. Data were normalized to untreated mice. Theresults in FIG. 23 showed the following: (1) miRNA inhibitors withcholesterol and limited phosphorothioate modifications could be used inin vivo experiments, and they induced robust inhibition of their targetsin the liver, upon systemic tail vein administration, in the absence ofany delivery reagents. Thus, the miRNA inhibitors showed the ability forself-delivery. In Example 20, the miRNA inhibitor-sterol conjugates areused for in vitro applications without delivery agents.

Example 20 In Vitro Use of miRNA Inhibitor-Sterol Conjugates

Sterol conjugates of the miRNA inhibitors, such as HP#81 with limitedphosphorothioate modifications and conjugated to cholesterol or anyother sterol can have cell-penetrating properties, i.e. they can be usedfor in vitro experiments without delivery (transfection) reagents. Suchsterol conjugates are used at 1 nanoMolar-10 microMolar concentrations,and, after incubation with the cells of interest, the amount of theoligonucleotide inside the cells is measured with Small RNA TAQMAN®assays (Cheng et al. (2009) Stem-loop RT-PCR quantification of siRNAs invitro and in vivo. Oligonucleotides, 19, 203-208) and biological effectssuch as upregulation of the mRNA targets-are measured by qRT-PCR at24-72 h post-transfection. Cells are typically maintained in theappropriate growth media containing up to 10% FBS, at 37° C., anddepending on the cell type, the density is usually between about1000-30,000 cells per well of a 96 well plate.

TABLE 1C Inhibitor Formats and Modifications loop posi- Linker LinkerComplementary Fig Seq ID tion HR I (L1) HR II HR III (L2) HR IV Targetsequence  3A SEQ ID NO: 22 HP01 5′ and GCUG AUCU CAGC GCUG AUCU CAGcmir 21 SEQ ID NO: 1 3′  3A SEQ ID NO: 23 HP02 5′ and GCUG L1 CAGC GCUGL1 CAGc mir 21 SEQ ID NO: 1 3′  3A SEQ ID NO: 24 HP03 5′ and GCUG L1CAGC GCUG L1 CAGc mir 21 SEQ ID NO: 2 3′  3A SEQ ID NO: 25 HP04 5′ GCUGAUCU CAGC na na na mir 21 SEQ ID NO: 3  3A SEQ ID NO: 26 HP05 5′ GCUG L1CAGC na na na mir 21 SEQ ID NO: 3  3A SEQ ID NO: 27 HP06 5′ GCUG L1 CAGCna na na mir 21 SEQ ID NO: 3  3A SEQ ID NO: 28 HP07 3′ na na na GCUGAUCU CAGc mir 21 SEQ ID NO: 1  3A SEQ ID NO: 29 HP08 3′ na na na GCUG L1CAGc mir 21 SEQ ID NO: 1  3A SEQ ID NO: 30 HP09 3′ na na na GCUG L1 CAGcmir 21 SEQ ID NO: 2  3A SEQ ID NO: 31 2'OMe none na na na na na namir 21 UCAACAUCAGUCUGAUAA GCUAN (SEQ ID NO: 31)  3B SEQ ID NO: 32 HP015′ and GCUG AUCU CAGC GCUG AUCU CAGc let7c SEQ ID NO: 4 3′  3BSEQ ID NO: 33 HP02 5′ and GCUG L1 CAGC GCUG L1 CAGc let7c SEQ ID NO: 43′  3B SEQ ID NO: 34 HP03 5′ and GCUG L1 CAGC GCUG L1 CAGc let7cSEQ ID NO: 5 3′  3B SEQ ID NO: 35 HP04 5′ GCUG AUCU CAGC na na na let7cSEQ ID NO: 6  3B SEQ ID NO: 36 HP05 5′ GCUG L1 CAGC na na na let7cSEQ ID NO: 6  3B SEQ ID NO: 37 HP06 5′ GCUG L1 CAGC na na na let7cSEQ ID NO: 6  3B SEQ ID NO: 38 HP07 3′ na na na GCUG AUCU CAGc let7cSEQ ID NO: 4  3B SEQ ID NO: 39 HP08 3′ na na na GCUG L1 CAGc let7cSEQ ID NO: 4  3B SEQ ID NO: 40 HP09 3′ na na na GCUG L1 CAGc let7cSEQ ID NO: 5  3B SEQ ID NO: 41 2′OMe none na na na na na na let7cAACCAUACAACCUACUAC CUCAN (SEQ ID NO: 41)  4A SEQ ID NO: 26 HP05 5′ GCUCL1 CAGC na na na mir 21 SEQ ID NO: 3  4A SEQ ID NO: 29 HP08 3′ na na naGCUG L1 CAGc mir 21 SEQ ID NO: 1  4A SEQ ID NO: 42 HP10 5′ GCG L1 CGC nana na mir 21 SEQ ID NO: 3  4A SEQ ID NO: 43 HP11 5′ GCGUG L1 CACGC na nana mir 21 SEQ ID NO: 3  4A SEQ ID NO: 44 HP12 5′ UGGC L1 CAGC na na namir 21 SEQ ID NO: 3  4A SEQ ID NO: 45 HP13 5′ GCUG AUUCU CAGC na na namir 21 SEQ ID NO: 3  4A SEQ ID NO: 46 HP14 3′ na na na GCG L1 CGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 47 HP15 3′ na na na GCGUG L1 CACGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 48 HP16 3′ na na na UGGC L1 CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 49 HP17 3′ na na na GCUG AUUCU CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 50 HP18 5′ GCUG L2 CAGC na na na mir 21SEQ ID NO: 3  4A SEQ ID NO: 51 HP19 5′ GCUG L3 CAGC na na na mir 21SEQ ID NO: 3  4A SEQ ID NO: 52 HP20 5′ GCUG L4 CAGC na na na mir 21SEQ ID NO: 3  4A SEQ ID NO: 53 HP21 5′ GCUG L5 CAGC na na na mir 21SEQ ID NO: 3  4A SEQ ID NO: 54 HP22 5′ GCUG L6 CAGC na na na mir 21SEQ ID NO: 3  4A SEQ ID NO: 55 HP23 5′ GCUG L7 CAGC na na na mir 21SEQ ID NO: 3  4A SEQ ID NO: 56 HP24 5′ GCUG L8 CAGC na na na mir 21SEQ ID NO: 3  4A SEQ ID NO: 57 HP25 3′ na na na GCUG L2 CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 58 HP26 3′ na na na GCUG L3 CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 59 HP27 3′ na na na GCUG L4 CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 60 HP28 3′ na na na GCUG L5 CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 61 HP29 3′ na na na GCUG L6 CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 62 HP30 3′ na na na GCUG L7 CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 63 HP31 3′ na na na GCUG L8 CAGc mir 21SEQ ID NO: 1  4A SEQ ID NO: 31 2′OMe none na na na na na na mir 21UCAACAUCAGUCUGAUAA GCUAN (SEQ ID NO:31)  4A Y mir 21  4A X mir 21  4ASEQ ID NO: 64 Neg none na na na na na na neg AAGUGGAUAUUGUUGCCA UCAN  (SEQ ID NO: 64)  4B SEQ ID NO: 36 HP05 5′ GCUC TEG CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 39 HP08 3′ na na na GCUG TEG CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 65 HP10 5′ GCG L1 CGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 66 HP11 5′ GCGUG L1 CACGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 67 HP12 5′ UGGC L1 CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 68 HP13 5′ GCUG AUUCU CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 69 HP14 3′ na na na GCG L1 CGc let7cSEQ ID NO: 4  4B SEQ ID NO: 70 HP15 3′ na na na GCGUG L1 CACGc let7cSEQ ID NO: 4  4B SEQ ID NO: 71 HP16 3′ na na na UGGC L1 CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 72 HP17 3′ na na na GCUG AUUCU CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 73 HP18 5′ GCUG L2 CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 74 HP19 5′ GCUG L3 CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 75 HP20 5′ GCUG L4 CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 76 HP21 5′ GCUG L5 CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 77 HP22 5′ GCUG L6 CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 78 HP23 5′ GCUG L7 CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 79 HP24 5′ GCUG L8 CAGC na na na let7cSEQ ID NO: 6  4B SEQ ID NO: 80 HP25 3′ na na na GCUG L2 CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 81 HP26 3′ na na na GCUG L3 CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 82 HP27 3′ na na na GCUG L4 CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 83 HP28 3′ na na na GCUG L5 CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 84 HP29 3′ na na na GCUG L6 CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 85 HP30 3′ na na na GCUG L7 CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 86 HP31 3′ na na na GCUG L8 CAGc let7cSEQ ID NO: 4  4B SEQ ID NO: 41 2′OMe none na na na na na na let7cAACCAUACAACCUACUAC CUCAN (SEQ ID NO: 41)  4B Y let7c  4B X let7c  4BSEQ ID NO: 64 neg none na na na na na na neg AAGUGGAUAUUGUUGCC AUCAN(SEQ ID NO: 64)  5 SEQ ID NO: 36 HP05 5′ GCUC TEG CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 39 HP08 3′ na na na GCUG TEG CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 65 HP10 5′ GCG L1 CGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 66 HP11 5′ GCGUG L1 CACGC na na na let7cSEQ ID NO: 6  5  SEQ ID NO: 67 HP12 5′ UGGC L1 CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 68 HP13 5′ GCUG AUUCU CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 69 HP14 3′ na na na GCG L1 CGc let7cSEQ ID NO: 4  5 SEQ ID NO: 70 HP15 3′ na na na GCGUG L1 CACGc let7cSEQ ID NO: 4  5 SEQ ID NO: 71 HP16 3′ na na na UGGC L1 CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 72 HP17 3′ na na na GCUG AUUCU CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 73 HP18 5′ GCUG L2 CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 74 HP19 5′ GCUG L3 CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 75 HP20 5′ GCUG L4 CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 76 HP21 5′ GCUG L5 CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 77 HP22 5′ GCUG L6 CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 78 HP23 5′ GCUG L7 CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 79 HP24 5′ GCUG L8 CAGC na na na let7cSEQ ID NO: 6  5 SEQ ID NO: 80 HP25 3′ na na na GCUG L2 CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 81 HP26 3′ na na na GCUG L3 CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 82 HP27 3′ na na na GCUG L4 CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 83 HP28 3′ na na na GCUG L5 CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 84 HP29 3′ na na na GCUG L6 CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 85 HP30 3′ na na na GCUG L7 CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 86 HP31 3′ na na na GCUG L8 CAGc let7cSEQ ID NO: 4  5 SEQ ID NO: 41 2′OMe none na na na na na na let7cAACCAUACAACCUACUAC CUCAN (SEQ ID NO: 41)  5 Y let7c  5 X let7c  5SEQ ID NO: 32 HP01 5′ and GCUG AUCU CAGC GCUG AUCU CAGc let7cSEQ ID NO: 4 3′  5 SEQ ID NO: 33 HP02 5′ and GCUG TEG CAGC GCUG AUCUCAGc let7c SEQ ID NO: 4   3′  5 SEQ ID NO: 64 neg none na na na na na naneg AAGUGGAUAUUGUUGCCA UCAN (SEQ ID NO: 64)  6A SEQ ID NO: 41 2′OMe nonena na na na na na let7c AACCAUACAACCUACUAC CUCAN (SEQ ID NO: 41)  6A Ylet7c  6A X let7c  6A SEQ ID NO: 79 HP24 5′ GCUG L8 CAGC na na na let7cSEQ ID NO: 6  6A NT let7c  6A SEQ ID NO: 64 Neg none na na na na na naneg AAGUGGAUAUUGUUGCCA UCAN (SEQ ID NO: 64)  6B SEQ ID NO: 31 2′OMe nonena na na na na na mir 21 UCAACAUCAGUCUGAUAA GCUAN (SEQ ID NO: 31)  6B Ymir 21  6B X mir 21  6B SEQ ID NO: 56 HP24 5′ GCUG L8 CAGC na na namir 21 SEQ ID NO: 3  6B NT mir 21  6B SEQ ID NO: 64 Neg none na na na nana na neg AAGUGGAUAUUGUUGCCA UCAN (SEQ ID NO: 64)  7A SEQ ID NO: 87 HP795′ GGCACG L3 CGUGCCAU na na na mir 21 SEQ ID NO: 3  7A SEQ ID NO: 88HP80 5′ GGCACG L3 CGUGCCA na na na mir 21 SEQ ID NO: 3  7A SEQ ID NO: 89HP81 5′ GGCACG L3 CGUGCC na na na mir 21 SEQ ID NO: 3  7A SEQ ID NO: 90HP82 3′ na na na UCCGUGC L3 GCACGGN mir 21 SEQ ID NO: 1  7ASEQ ID NO: 91 HP83 3′ na na na UCCGUGC L3 GCACGg mir 21 SEQ ID NO: 1  7ASEQ ID NO: 31 2′OMe none na na na na na na mir 21 UCAACAUCAGUCUGAUAAGCUAN (SEQ ID NO: 31)  7A Y mir 21  7A X mir 21  7B SEQ ID NO: 92 HP795′ GGCACG L3 CGUGCCAU na na na let7a SEQ ID NO: 7  7B SEQ ID NO: 93 HP805′ GGCACG L3 CGUGCCA na na na let7a SEQ ID NO: 7  7B SEQ ID NO: 94 HP815′ GGCACG L3 CGUGCC na na na let7a SEQ ID NO: 7  7B SEQ ID NO: 95 HP823′ na na na UCCGUGC L3 GCACGGN let7a SEQ ID NO: 8  7B SEQ ID NO: 96 HP833′ na na na UCCGUGC L3 GCACGg let7a SEQ ID NO: 8  7B SEQ ID NO: 97 2′OMenone na na na na na na let7a AACUAUACAACCUACUAC CUCAN (SEQ ID NO: 97) 7B Y let7a  7B X let7a  7C SEQ ID NO: 98 HP79 5′ GGCACG L3 CGUGCCAU nana na let7c SEQ ID NO: 9  7C SEQ ID NO: 99 HP80 5′ GGCACG L3 CGUGCCA nana na let7c SEQ ID NO: 9  7C SEQ ID NO: 100 HP81 5′ GGCACG L3 CGUGCC nana na let7c SEQ ID NO: 9  7C SEQ ID NO: 101 HP82 3′ na na na UCCGUGC L3GCACGGN let7c SEQ ID NO: 4  7C SEQ ID NO: 102 HP83 3′ na na na UCCGUGCL3 GCACGg let7c SEQ ID NO: 4  7C SEQ ID NO: 41 2′OMe none na na na na nana let7c AACCAUACAACCUACUAC CUCAN (SEQ ID NO: 41)  7C Y let7c  7C Xlet7c  8A SEQ ID NO: 103 BTM03 na na na na na na na mir21 U_(f)C_(f)AAC_(f)AU_(f)C_(f) AGU_(f)C_(f) U_(f) GAU_(f) AAGC_(f)U_(f)A(SEQ ID NO: 153)  8A SEQ ID NO: 104 BTM04 na na na na na na na mir21 U_(f)C_(f) AAC_(f) AU_(f)C_(f) AGU_(f)C_(f) U_(f) GAU_(f)(SEQ ID NO: 104)  8A SEQ ID NO: 105 BTM05 na na na na na na na mir21 TCAA C A TC AG TCT GA T AA G CT A (SEQ ID NO: 105)  8A SEQ ID NO: 106BTM06 na na na na na na na mir21 TC AA C A TC AG TCT GA T(SEQ ID NO: 155)  8A SEQ ID NO: 107 BTM07 na na na na na na na mir21 U_(f)C_(f) AAC_(f) AU_(f)C_(f) AGU_(f)C_(f) U_(f) GAU_(f) AAGC_(f)U_(f)A (SEQ ID NO: 107)  8A SEQ ID NO: 108 BTM22 na na na na na na na mir21 U_(f)C_(f) AAC_(f) AU_(f)C_(f) AGU_(f)C_(f) U_(f) GAU_(f) AAGC_(f)U_(f)AN (SEQ ID NO: 108)  8A SEQ ID NO: 31 2′OMe none na na na na na na mir21UCAACAUCAGUCUGAUAA GCUAN (SEQ ID NO: 31)  8A X mir21  8A SEQ ID NO: 64Neg neg AAGUGGAUAUUGUUGCCA UCAN (SEQ ID NO: 64)  8B SEQ ID NO: 109 BTM03na na na na na na na let7a AAC_(f)U_(f) AU_(f) AC_(f) AAC_(f)C_(f) U_(f)AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) A (SEQ ID NO: 109)  8B SEQ ID NO: 110BTM04 na na na na na na na let7a AAC_(f)U_(f) AU_(f) AC_(f) AAC_(f)C_(f)U_(f) AC_(f)U_(f) (SEQ ID NO: 110)  8B SEQ ID NO: 111 BTM05 na na na nana na na let7a AA CT A T A C AA CCT A CT A C CTC A (SEQ ID NO: 111)  8BSEQ ID NO: 112 BTM06 na na na na na na na let7a AA CT A T A C AA CCT ACT (SEQ ID NO: 112)  8B SEQ ID NO: 113 BTM07 na na na na na na na let7aAAC_(f)U_(f) AU_(f) AC_(f) AAC_(f)C_(f) U_(f) AC_(f)U_(f)AC_(f)C_(f)U_(f)C_(f) A (SEQ ID NO: 113)  8B SEQ ID NO: 114 BTM22 na nana na na na na let7a AAC_(f)U_(f) AU_(f) AC_(f) AAC_(f)C_(f) U_(f)AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) AN (SEQ ID NO: 114)  8B SEQ ID NO: 972′OMe none na na na na na na let7a AACUAUACAACCUACUAC CUCAN(SEQ ID NO: 97)  8B X let7a  8B SEQ ID NO: 64 Neg neg AAGUGGAUAUUGUUGCCAUCAN (SEQ ID NO: 64)  8C SEQ ID NO: 115 BTM03 na na na na na na na let7cAAC_(f)C_(f) AU_(f) AC_(f) AAC_(f)C_(f) U_(f) AC_(f)U_(f)AC_(f)C_(f)U_(f)C_(f) A (SEQ ID NO: 115)  8C SEQ ID NO: 116 BTM04 na nana na na na na let7c AAC_(f)C_(f) AU_(f) AC_(f) AAC_(f)C_(f) U_(f)AC_(f)U_(f) (SEQ ID NO: 116)  8C SEQ ID NO: 117 BTM05 na na na na na nana let7c AA CC A T A C AA CCT A CT A C CTC A (SEQ ID NO: 117)  8CSEQ ID NO: 118 BTM06 na na na na na na na let7c AA CC A T A C AA CCT ACT (SEQ ID NO: 118)  8C SEQ ID NO: 119 BTM07 na na na na na na na let7cAAC_(f)C_(f) AU_(f) AC_(f) AAC_(f)C_(f) U_(f) AC_(f)U_(f)AC_(f)C_(f)U_(f)C_(f) A (SEQ ID NO: 119)  8C SEQ ID NO: 120 BTM22 na nana na na na na let7c AAC_(f)C_(f) AU_(f) AC_(f) AAC_(f)C_(f) U_(f)AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) AN (SEQ ID NO: 120)  8C SEQ ID NO: 412′OMe none na na na na na na let7c AACCAUACAACCUACUAC CUCAN(SEQ ID NO: 41)  8C X let7c  8C SEQ ID NO: 64 Neg neg AAGUGGAUAUUGUUGCCAUCAN (SEQ ID NO: 64) 12 SEQ ID NO: 120 HP98 5′ GGCACG L3 CGUGCC na na namir122 SEQ ID NO: 10 12 SEQ ID NO: 121 HP101 3′ na na na UCCGUGC L3GCACGGN mir122 SEQ ID NO: 11 12 SEQ ID NO: 122 HP81 5′ GGCACG L3 CGUGCCna na na mir122 SEQ ID NO: 12 12 SEQ ID NO: 123 HP98 5′ GGCACG L3 CGUGCCna na na let7c SEQ ID NO: 13 13 SEQ ID NO: 124 HP79 5′ GGCACG L3CGUGCCAU na na na let7a SEQ ID NO: 7 13 SEQ ID NO: 125 HP85 5′ GGCACGL11 CGUGCC na na na let7a SEQ ID NO: 7 13 SEQ ID NO: 126 HP88 5′ GGCACGL3 CGUGCC na na na let7a SEQ ID NO: 14 13 SEQ ID NO: 127 HP93 5′ GGCACGL10 CGUGCC na na na let7a SEQ ID NO: 7 13 SEQ ID NO: 128 HP96 5′ GGCACGL3 CGUGCC na na na let7a SEQ ID NO: 15 13 SEQ ID NO: 97 2′OMe none na nana na na na let7a AACUAUACAACCUACUAC CUCAN (SEQ ID NO: 97) 13 Y let7a 13X let7a 14 SEQ ID NO: 129 HP81 5′ GGCACG L3 CGUGCC na na na mir 21SEQ ID NO: 3 14 SEQ ID NO: 130 HP85 5′ GGCACG L11 CGUGCC na na na mir 21SEQ ID NO: 3 14 SEQ ID NO: 131 HP88 5′ GGCACG L3 CGUGCC na na na mir 21SEQ ID NO: 16 14 SEQ ID NO: 132 HP93 5′ GGCACG L10 CGUGCC na na namir 21 SEQ ID NO: 3 14 SEQ ID NO: 133 HP96 5′ GGCACG L3 CGUGCC na na namir 21 SEQ ID NO: 17 14 SEQ ID NO: 31 2′OMe none na na na na na namir 21 UCAACAUCAGUCUGAUAA GCUAN (SEQ ID NO: 31) 14 X mir 21 14 Y mir 2115 SEQ ID NO: 97 2′OMe none na na na na na na let7a AACUAUACAACCUACUACCUCAN (SEQ ID NO: 97) 15 X 15 Y 15 SEQ ID NO: 134 HP81 5′ GGCACG L3CGUGCC na na na let7a SEQ ID NO: 7 15 SEQ ID NO: 135 HP89 5′ GGCACG L3CGUGCC na na na let7a SEQ ID NO: 18 15 SEQ ID NO: 136 HP90 5′ GGCACG L3CGUGCC na na na let7a SEQ ID NO: 19 15 SEQ ID NO: 137 HP91 5′ GGCACG L3CGUGCC na na na let7a SEQ ID NO: 20 15 SEQ ID NO: 138 HP92 5′ GGCACG L3CGUGCC na na na let7a SEQ ID NO: 21

TABLE 1D miRNA FIG. Sequence Identifier Inhibitor sequence inhibitor  3ASEQ ID NO: 22 GCUGAUCUCAGCUCAACAUCAGUCUGAUAAGCUAGCUGAUCU HP01 CAGc  3ASEQ ID NO: 23 GCUG-L1-CAGCUCAACAUCAGUCUGAUAAGCUAGCUG-L1- HP02 CAGc  3ASEQ ID NO: 24 GCUG-L1-CAGCUCAACAUCAGUCUGAUAAGCUAGCUG-L1- HP03 CAGc  3ASEQ ID NO: 25 GCUGAUCUCAGCUCAACAUCAGUCUGAUAAGCUAc HP04  3A SEQ ID NO: 26GCUG-L1-CAGCUCAACAUCAGUCUGAUAAGCUAc HP05  3A SEQ ID NO: 27GCUG-L1-CAGCUCAACAUCAGUCUGAUAAGCUAc HP06  3A SEQ ID NO: 28UCAACAUCAGUCUGAUAAGCUAGCUGAUCUCAGc HP07  3A SEQ ID NO: 29UCAACAUCAGUCUGAUAAGCUAGCUG-L1-CAGc HP08  3A SEQ ID NO: 30UCAACAUCAGUCUGAUAAGCUAGCUG-L1-CAGc HP09  3A SEQ ID NO: 31UCAACAUCAGUCUGAUAAGCUAN 2′OMe  3B SEQ ID NO: 32GCUGAUCUCAGCAACCAUACAACCUACUACCUCAGCUGAUCU HP01 CAGc  3B SEQ ID NO: 33GCUG-L1-CAGCAACCAUACAACCUACUACCUCAGCUG-L1- HP02 CAGc  3B SEQ ID NO:34GCUG-L1-CAGCAACCAUACAACCUACUACCUCAGCUG-L1- HP03 CAGc  3B SEQ ID NO: 35GCUGAUCUCAGCAACCAUACAACCUACUACCUCAc HP04  3B SEQ ID NO: 36GCUG-L1-CAGCAACCAUACAACCUACUACCUCAc HP05  3B SEQ ID NO: 37GCUG-L1-CAGCAACCAUACAACCUACUACCUCAc HP06  3B SEQ ID NO: 38AACCAUACAACCUACUACCUCAGCUGAUCUCAGc HP07  3B SEQ ID NO: 39AACCAUACAACCUACUACCUCAGCUG-L1-CAGc HP08  3B SEQ ID NO: 40AACCAUACAACCUACUACCUCAGCUG-L1-CAGc HP09  3B SEQ ID NO: 41AACCAUACAACCUACUACCUCAN 2′OMe  4A SEQ ID NO: 158GCUC-L1-CAGCUCAACAUCAGUCUGAUAAGCUAc HP05  4A SEQ ID NO: 29UCAACAUCAGUCUGAUAAGCUAGCUG-L1-CAGc HP08  4A SEQ ID NO: 42GCG-L1-CGCUCAACAUCAGUCUGAUAAGCUAc HP10  4A SEQ ID NO: 43GCGUG-L1-CACGCUCAACAUCAGUCUGAUAAGCUAc HP11  4A SEQ ID NO: 44UGGC-L1-CAGCUCAACAUCAGUCUGAUAAGCUAc HP12  4A SEQ ID NO: 45GCUGAUUCUCAGCUCAACAUCAGUCUGAUAAGCUAc HP13  4A SEQ ID NO: 46UCAACAUCAGUCUGAUAAGCUAGCG-L1-CGc HP14  4A SEQ ID NO: 47UCAACAUCAGUCUGAUAAGCUAGCGUG-L1-CACGc HP15  4A SEQ ID NO: 48UCAACAUCAGUCUGAUAAGCUAUGGC-L1-CAGc HP16  4A SEQ ID NO: 49UCAACAUCAGUCUGAUAAGCUAGCUGAUUCUCAGc HP17  4A SEQ ID NO: 50GCUG-L2-CAGCUCAACAUCAGUCUGAUAAGCUAc HP18  4A SEQ ID NO: 51GCUG-L3-CAGCUCAACAUCAGUCUGAUAAGCUAc HP19  4A SEQ ID NO: 52GCUG-L4-CAGCUCAACAUCAGUCUGAUAAGCUAc HP20  4A SEQ ID NO: 53GCUG-L5-CAGCUCAACAUCAGUCUGAUAAGCUAc HP21  4A SEQ ID NO: 54GCUG-L6-CAGCUCAACAUCAGUCUGAUAAGCUAc HP22  4A SEQ ID NO: 55GCUG-L7-CAGCUCAACAUCAGUCUGAUAAGCUAc HP23  4A SEQ ID NO: 56GCUG-L8-CAGCUCAACAUCAGUCUGAUAAGCUAc HP24  4A SEQ ID NO: 57UCAACAUCAGUCUGAUAAGCUAGCUG-L2-CAGc HP25  4A SEQ ID NO: 58UCAACAUCAGUCUGAUAAGCUAGCUG-L3-CAGc HP26  4A SEQ ID NO: 59UCAACAUCAGUCUGAUAAGCUAGCUG-L4-CAGc HP27  4A SEQ ID NO: 60UCAACAUCAGUCUGAUAAGCUAGCUG-L5-CAGc HP28  4A SEQ ID NO: 61UCAACAUCAGUCUGAUAAGCUAGCUG-L6-CAGc HP29  4A SEQ ID NO: 62UCAACAUCAGUCUGAUAAGCUAGCUG-L7-CAGc HP30  4A SEQ ID NO: 63UCAACAUCAGUCUGAUAAGCUAGCUG-L8-CAGc HP31  4A SEQ ID NO: 31UCAACAUCAGUCUGAUAAGCUAN 2′OMe  4A SEQ ID NO: 64 AAGUGGAUAUUGUUGCCAUCANNeg  4B SEQ ID NO: 160 GCUC-L1-CAGCAACCAUACAACCUACUACCUCAc HP05  4BSEQ ID NO: 39 AACCAUACAACCUACUACCUCAGCUG-L1-CAGc HP08  4B SEQ ID NO: 65GCG-L1-CGCAACCAUACAACCUACUACCUCAc HP10  4B SEQ ID NO: 66GCGUG-L1-CACGCAACCAUACAACCUACUACCUCAc HP11  4B SEQ ID NO: 67UGGC-L1-CAGCAACCAUACAACCUACUACCUCAc HP12  4B SEQ ID NO: 68GCUGAUUCUCAGCAACCAUACAACCUACUACCUCAc HP13  4B SEQ ID NO: 69AACCAUACAACCUACUACCUCAGCG-L1-CGc HP14  4B SEQ ID NO: 70AACCAUACAACCUACUACCUCAGCGUG-L1-CACGc HP15  4B SEQ ID NO: 71AACCAUACAACCUACUACCUCAUGGC-L1-CAGc HP16  4B SEQ ID NO: 72AACCAUACAACCUACUACCUCAGCUGAUUCUCAGc HP17  4B SEQ ID NO: 73GCUG-L2-CAGCAACCAUACAACCUACUACCUCAc HP18  4B SEQ ID NO: 74GCUG-L3-CAGCAACCAUACAACCUACUACCUCAc HP19  4B SEQ ID NO: 75GCUG-L4-CAGCAACCAUACAACCUACUACCUCAc HP20  4B SEQ ID NO: 76GCUG-L5-CAGCAACCAUACAACCUACUACCUCAc HP21  4B SEQ ID NO: 77GCUG-L6-CAGCAACCAUACAACCUACUACCUCAc HP22  4B SEQ ID NO: 78GCUG-L7-CAGCAACCAUACAACCUACUACCUCAc HP23  4B SEQ ID NO: 79GCUG-L8-CAGCAACCAUACAACCUACUACCUCAc HP24  4B SEQ ID NO: 80AACCAUACAACCUACUACCUCAGCUG-L2-CAGc HP25  4B SEQ ID NO: 81AACCAUACAACCUACUACCUCAGCUG-L3-CAGc HP26  4B SEQ ID NO: 82AACCAUACAACCUACUACCUCAGCUG-L4-CAGc HP27  4B SEQ ID NO: 83AACCAUACAACCUACUACCUCAGCUG-L5-CAGc HP28  4B SEQ ID NO: 84AACCAUACAACCUACUACCUCAGCUG-L6-CAGc HP29  4B SEQ ID NO: 85AACCAUACAACCUACUACCUCAGCUG-L7-CAGc HP30  4B SEQ ID NO: 86AACCAUACAACCUACUACCUCAGCUG-L8-CAGc HP31  4B SEQ ID NO: 41AACCAUACAACCUACUACCUCAN 2′OMe  4B SEQ ID NO: 64 AAGUGGAUAUUGUUGCCAUCANneg  5 SEQ ID NO: 36 GCUC-L1-CAGCAACCAUACAACCUACUACCUCAc HP05  5SEQ ID NO: 39 AACCAUACAACCUACUACCUCAGCUG-L1-CAGc HP08  5  SEQ ID NO: 65GCG-L1-CGCAACCAUACAACCUACUACCUCAc HP10  5 SEQ ID NO: 66GCGUG-L1-CACGCAACCAUACAACCUACUACCUCAc HP11  5 SEQ ID NO: 67UGGC-L1-CAGCAACCAUACAACCUACUACCUCAc HP12  5 SEQ ID NO: 68GCUGAUUCUCAGCAACCAUACAACCUACUACCUCAc HP13  5 SEQ ID NO: 69AACCAUACAACCUACUACCUCAGCG-L1-CGc HP14  5 SEQ ID NO: 70AACCAUACAACCUACUACCUCAGCGUG-L1-CACGc HP15  5 SEQ ID NO: 71AACCAUACAACCUACUACCUCAUGGC-L1-CAGc HP16  5 SEQ ID NO: 72AACCAUACAACCUACUACCUCAGCUGAUUCUCAGc HP17  5 SEQ ID NO: 73GCUG-L2-CAGCAACCAUACAACCUACUACCUCAc HP18  5 SEQ ID NO: 74GCUG-L3-CAGCAACCAUACAACCUACUACCUCAc HP19  5 SEQ ID NO: 75GCUG-L4-CAGCAACCAUACAACCUACUACCUCAc HP20  5 SEQ ID NO: 76GCUG-L5-CAGCAACCAUACAACCUACUACCUCAc HP21  5 SEQ ID NO: 77GCUG-L6-CAGCAACCAUACAACCUACUACCUCAc HP22  5 SEQ ID NO: 78GCUG-L7-CAGCAACCAUACAACCUACUACCUCAc HP23  5  SEQ ID NO: 79GCUG-L8-CAGCAACCAUACAACCUACUACCUCAc HP24  5 SEQ ID NO: 80AACCAUACAACCUACUACCUCAGCUG-L2-CAGc HP25  5 SEQ ID NO: 81AACCAUACAACCUACUACCUCAGCUG-L3-CAGc HP26  5 SEQ ID NO: 82AACCAUACAACCUACUACCUCAGCUG-L4-CAGc HP27  5 SEQ ID NO: 83AACCAUACAACCUACUACCUCAGCUG-L5-CAGc HP28  5 SEQ ID NO: 84AACCAUACAACCUACUACCUCAGCUG-L6-CAGc HP29  5 SEQ ID NO: 85AACCAUACAACCUACUACCUCAGCUG-L7-CAGc HP30  5 SEQ ID NO: 86AACCAUACAACCUACUACCUCAGCUG-L8-CAGc HP31  5 SEQ ID NO: 41AACCAUACAACCUACUACCUCAN 2′OMe  5 SEQ ID NO: 32GCUGAUCUCAGCAACCAUACAACCUACUACCUCAGCUGAUCU HP01   CAGc  5 SEQ ID NO: 159GCUG-L1-CAGCAACCAUACAACCUACUACCUCAGCUGAUCU HP02 CAGc  5 SEQ ID NO: 64AAGUGGAUAUUGUUGCCAUCAN neg  6A SEQ ID NO: 41 AACCAUACAACCUACUACCUCAN2′OMe  6A SEQ ID NO: 79 GCUG-L8-CAGCAACCAUACAACCUACUACCUCAc HP24  6ASEQ ID NO: 64 AAGUGGAUAUUGUUGCCAUCAN Neg  6B SEQ ID NO: 31UCAACAUCAGUCUGAUAAGCUAN 2′OMe  6B SEQ ID NO: 56GCUG-L8-CAGCUCAACAUCAGUCUGAUAAGCUAc HP24  6B SEQ ID NO: 64AAGUGGAUAUUGUUGCCAUCAN Neg  7A SEQ ID NO: 87GGCACG-L3-CGUGCCAUUCAACAUCAGUCUGAUAAGCUAc HP79  7A SEQ ID NO: 88GGCACG-L3-CGUGCCAUCAACAUCAGUCUGAUAAGCUAc HP80  7A SEQ ID NO: 89GGCACG-L3-CGUGCCUCAACAUCAGUCUGAUAAGCUAc HP81  7A SEQ ID NO: 90UCAACAUCAGUCUGAUAAGCUAUCCGUGC-L3-GCACGGN HP82  7A SEQ ID NO: 91UCAACAUCAGUCUGAUAAGCUAUCCGUGC-L3-GCACGg HP83  7A SEQ ID NO: 31UCAACAUCAGUCUGAUAAGCUAN 2′OMe  7B SEQ ID NO: 92GGCACG-L3-CGUGCCAUAACUAUACAACCUACUACCUCAt HP79  7B SEQ ID NO: 93GGCACG-L3-CGUGCCAAACUAUACAACCUACUACCUCAt HP80  7B SEQ ID NO: 94GGCACG-L3-CGUGCCAACUAUACAACCUACUACCUCAt HP81  7B SEQ ID NO: 95AACUAUACAACCUACUACCUCAUCCGUGC-L3-GCACGGN HP82  7B SEQ ID NO: 96AACUAUACAACCUACUACCUCAUCCGUGC-L3-GCACGg HP83  7B SEQ ID NO: 97AACUAUACAACCUACUACCUCAN 2′OMe  7C SEQ ID NO: 98GGCACG-L3-CGUGCCAUAACCAUACAACCUACUACCUCAg HP79  7C SEQ ID NO: 99GGCACG-L3-CGUGCCAAACCAUACAACCUACUACCUCAg HP80  7C SEQ ID NO: 100GGCACG-L3-CGUGCCAACCAUACAACCUACUACCUCAg HP81  7C SEQ ID NO: 101AACCAUACAACCUACUACCUCAUCCGUGC-L3-GCACGGN HP82  7C SEQ ID NO: 102AACCAUACAACCUACUACCUCAUCCGUGC-L3-GCACGg HP83  7C SEQ ID NO: 41AACCAUACAACCUACUACCUCAN 2′OMe  8A SEQ ID NO: 153 U_(f)C_(f) AAC_(f)AU_(f)C_(f) AGU_(f)C_(f)U_(f) GAU_(f) AAGC_(f)U_(f) A BTM03  8ASEQ ID NO: 104 U_(f)C_(f) AAC_(f) AU_(f)C_(f) AGU_(f)C_(f)U_(f) GAU_(f)BTM04  8A SEQ ID NO: 105 TC AA C A TC AG TCT GA T AAG CT A BTM05  8ASEQ ID NO: 155 TC AA C A TC AG TCT GA T BTM06  8A SEQ ID NO: 107U_(f)C_(f) AAC_(f) AU_(f)C_(f) AGU_(f)C_(f)U_(f) GAU_(f) AAGC_(f)U_(f) ABTM07  8A SEQ ID NO: 108 U_(f)C_(f) AAC_(f) AU_(f)C_(f)AGU_(f)C_(f)U_(f) GAU_(f) AAGC_(f)U_(f) AN BTM22  8A SEQ ID NO: 31UCAACAUCAGUCUGAUAAGCUAN 2′OMe  8A SEQ ID NO: 64 AAGUGGAUAUUGUUGCCAUCANNeg  8B SEQ ID NO: 109 AAC_(f)U_(f) AU_(f) AC_(f) AAC_(f)C_(f)U_(f)AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) A BTM03  8B SEQ ID NO: 110AAC_(f)U_(f) AU_(f) AC_(f) AAC_(f)C_(f)U_(f) AC_(f)U_(f) BTM04  8BSEQ ID NO: 111 AA CT A T A C AA CCT A CT A CCTC A BTM05  8BSEQ ID NO: 112 AA CT A T A C AA CCT A CT BTM06  8B SEQ ID NO: 113AAC_(f)U_(f) AU_(f) AC_(f) AAC_(f)C_(f)U_(f) AC_(f)U_(f)AC_(f)C_(f)U_(f)C_(f) A BTM07  8B SEQ ID NO: 114 AAC_(f)U_(f) AU_(f)AC_(f) AAC_(f)C_(f)U_(f) AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) AN BTM22  8BSEQ ID NO: 97 AACUAUACAACCUACUACCUCAN 2′OMe  8B SEQ ID NO: 64AAGUGGAUAUUGUUGCCAUCAN Neg  8C SEQ ID NO: 115 AAC_(f)C_(f) AU_(f) AC_(f)AAC_(f)C_(f)U_(f) AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) A BTM03  8CSEQ ID NO: 116 AAC_(f)C_(f) AU_(f) AC_(f) AAC_(f)C_(f)U_(f) AC_(f)U_(f)BTM04  8C SEQ ID NO: 117 AA CC A T A C AA CCT A CT A CCTC A BTM05  8CSEQ ID NO: 118 AA CC A T A C AA CCT A CT BTM06  8C SEQ ID NO: 119AAC_(f)C_(f) AU_(f) AC_(f) AAC_(f)C_(f)U_(f) AC_(f)U_(f)AC_(f)C_(f)U_(f)C_(f) A BTM07  8C SEQ ID NO: 120 AAC_(f)C_(f) AU_(f)AC_(f) AAC_(f)C_(f)U_(f) AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) AN BTM22  8CSEQ ID NO: 41 AACCAUACAACCUACUACCUCAN 2′OMe  8C SEQ ID NO: 64AAGUGGAUAUUGUUGCCAUCAN Neg 12 SEQ ID NO: 161 GGCACG-L3-CGUGCCACAAACACCAUUGUCACACUCCA c HP98 12 SEQ ID NO: 121 ACAAACACCAUUGUCACACUCCAUCCGUGC-L3-GCACGGN HP101 12 SEQ ID NO: 122GGCACG-L3-CGUGCCACAAACACCAUUGUCACACUCCAc HP81 12 SEQ ID NO: 123GGCACG-L3-CGUGCC AACCAUACAACCUACUACCUCA g HP98 13 SEQ ID NO: 124GGCACG-L3-CGUGCCAUAACUAUACAACCUACUACCUCAt HP79 13 SEQ ID NO: 125GGCACG-L11-CGUGCCAACUAUACAACCUACUACCUCAt HP85 13 SEQ ID NO: 126GGCACG-L3-CGUGCCAACUAUACAACCUACUACCUCAY HP88 13 SEQ ID NO: 127GGCACG-L10-CGUGCCAACUAUACAACCUACUACCUCAt HP93 13 SEQ ID NO: 128GGCACG-L3-CGUGCCAACUAUACAACCUACUACCUCA-Chol HP96 13 SEQ ID NO: 97AACUAUACAACCUACUACCUCAN 2′OMe 14 SEQ ID NO: 129GGCACG-L3-CGUGCCUCAACAUCAGUCUGAUAAGCUAc HP81 14 SEQ ID NO: 130GGCACG-L11-CGUGCCUCAACAUCAGUCUGAUAAGCUAc HP85 14 SEQ ID NO: 131GGCACG-L3-CGUGCCUCAACAUCAGUCUGAUAAGCUAY HP88 14 SEQ ID NO: 132GGCACG-L10-CGUGCCUCAACAUCAGUCUGAUAAGCUAc HP93 14 SEQ ID NO: 133GGCACG-L3-CGUGCCUCAACAUCAGUCUGAUAAGCUA-Chol HP96 14 SEQ ID NO: 31UCAACAUCAGUCUGAUAAGCUAN 2′OMe 15 SEQ ID NO: 97 AACUAUACAACCUACUACCUCAN2′OMe 15 SEQ ID NO: 134 GGCACG-L3-CGUGCCAACUAUACAACCUACUACCUCAt HP81 15SEQ ID NO: 135 GGCACG-L3-CGUGCCAACUAUACAACCU A C T A C C T C At HP89 15SEQ ID NO: 136 GGCACG-L3-CGUGCCAA C U G U A C A AACUACUACCUCAt HP90 15SEQ ID NO: 137 GGCACG-L3-CGUGCCAAC_(f)U_(f) AU_(f) AC_(f)AAC_(f)C_(f)U_(f) AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) At HP91 15SEQ ID NO: 138 GGCACG-L3-CGUGCC AAC_(f)U_(f) AU_(f) AC_(f)AAC_(f)C_(f)U_(f) AC_(f)U_(f) AC_(f)C_(f)U_(f)C_(f) At HP92

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims. The entirecontents of all patents, published patent applications, and otherreferences cited herein are hereby expressly incorporated herein intheir entireties by reference.

1. A composite nucleic acid inhibitory molecule which comprises thefollowing regions: a. a first homologous region, b. a loop, c. a secondhomologous region, and d. a target binding nucleic acid segment which isgreater than 6 nucleotides in length, wherein the loop connects thefirst homologous region and the second homologous region and is ofsufficient length to allow for the first homologous region and thesecond homologous region to hybridize to one another to form adouble-stranded structure, and wherein the first homologous region andthe second homologous region share sufficient sequence homology to allowfor hybridization under physiological conditions.
 2. The compositenucleic acid inhibitory molecule of claim 1, wherein the target is along non-coding RNA or a short non-coding RNA.
 3. The composite nucleicacid inhibitory molecule of claim 2, wherein the short non-coding RNA ischosen from a microRNA (miRNA), a Piwi-interacting RNA (piRNA), and asmall interfering RNA (siRNA).
 4. The composite nucleic acid inhibitorymolecule of claim 1, wherein the loop comprises a non-nucleotide loop.5. The composite nucleic acid inhibitory molecule of claim 1, whereinthe target binding nucleic acid segment is about 60 to about 100%complementary to all or a portion of at least one target nucleic acid.6. The composite nucleic acid inhibitory molecule of claim 1, whereinthe first homologous region or the second homologous region is betweenfrom about 3 nucleotides to about 30 nucleotides in length.
 7. Thecomposite nucleic acid inhibitory molecule of claim 1, wherein the firsthomologous region is between about 4 and about 10 nucleotides in length.8. The composite nucleic acid inhibitory molecule of claim 3, whereinthe backbone of the loop comprises covalently bonded carbon atoms. 9.The composite nucleic acid inhibitory molecule of claim 4, wherein thebackbone of the loop comprises covalently bonded carbon and oxygenatoms.
 10. The composite nucleic acid inhibitory molecule of claim 1,wherein the loop is chosen from polyethylene glycol, C2-C18 alkane diol,styrene, stilbene, triazole, tetrazole, nucleic acid, poly abasicnucleoside, polysaccharide, peptide, polyamide, hydrazone, oxyimine,polyester, disulfide, polyamine, polyether, peptide nucleic acid,cycloalkane, polyalkene, aryl, derivatives thereof and any combinationthereof.
 11. The composite nucleic acid inhibitory molecule of claim 1,wherein the homologous region is separated from the target bindingnucleic acid segment by at least one nucleotide having a purine base, apyrimidine base, or no base (abasic).
 12. The composite nucleic acidinhibitory molecule of claim 1, wherein the nucleotides that comprisethe target-binding nucleic acid segment are modified and thosemodifications are chosen from, DNA, RNA, 2′OMe, 2′OAllyl, 2′O-propargyl,2′O-alkyl, 2′ fluoro, 2′ arabino, 2′ xylo, 2′ fluoro arabino,phosphorothioate, phosphorodithioate, phosphoroamidates, 2′ amino,5-alkyl-substituted pyrimidine, 5-halo-substituted pyrimidine,alkyl-substituted purine, halo-substituted purine, bicyclic nucleotides,2′MOE, LNA, LNA-like molecules and derivatives thereof.
 13. Thecomposite nucleic acid inhibitory molecule of claim 10, wherein thepolyethylene glycol is a polyethylene glycol derivative and wherein thepolyethylene glycol derivative is hexa-ethylene glycol or penta-ethyleneglycol.
 14. The composite nucleic acid inhibitory molecule of claim 9,wherein the loop is C12 alkane diol.
 15. The composite nucleic acidinhibitory molecule of claim 1, wherein the molecule is modified and themodification adds at least one amine, imine, guanidine, or aromaticamino heterocycle.
 16. The composite nucleic acid inhibitory molecule ofclaim 1, wherein the nucleotides in the homologous region and/or spacercomprises at least one 2′ O-alkyl, LNA, 2′ fluoro, arabino, 2′ xylo, 2′fluoro arabino, phosphorothioate, phosphorodithioate, 2′ amino, bicyclicnucleotide, 5-alkyl-substituted pyrimidine, 5-halo-substitutedpyrimidine, alkyl-substituted purine, or halo-substituted purine,halo-substituted purine, bicyclic nucleotides, 2′MOE, LNA, andderivatives thereof.
 17. The composite nucleic acid inhibitory moleculeof claim 1, wherein the target binding nucleic acid segment is betweenfrom about 10 nucleotides to about 100 nucleotides in length.
 18. Thecomposite nucleic acid inhibitory molecule of claim 1, wherein thetarget binding nucleic acid segment is between from about 15 to about 25nucleotides in length.
 19. The composite nucleic acid inhibitorymolecule of claim 1, wherein the first homologous region, the secondhomologous region and the loop form a stem loop structure and the stemloop structure is on the 5′ end of the nucleic acid inhibitory molecule.20. The composite nucleic acid inhibitory molecule of claim 1, whereinthe first homologous region, the second homologous region and the loopform a stem loop structure and the stem loop structure is on the 3′ endof the nucleic acid inhibitory molecule.
 21. The composite nucleic acidinhibitory molecule of claim 20, further comprising a second stem loopstructure at the 5′ end of the nucleic acid inhibitory molecule.
 22. Thecomposite nucleic acid inhibitory molecule of claim 1, wherein theentire molecule is between about 20 and about 150 nucleotides in length.23. The composite nucleic acid inhibitory molecule of claim 1, whereinthe inhibitory molecule is modified and the modification is a covalentlylinked conjugate that enhances cell penetration, endocytosis,facilitated diffusion, tissue localization, inhibitory moleculedetection, or cellular trafficking of the modified nucleic acidinhibitory molecule.
 24. The composite nucleic acid inhibitory moleculeof claim 1, wherein the molecule further comprises one or morenucleotides on either end of the molecule.
 25. A multimeric nucleic acidinhibitory molecule comprising at least a first oligonucleotide and asecond oligonucleotide, wherein the first oligonucleotide comprises atarget nucleic acid molecule binding region, and two regions which willnot hybridize to the target nucleic acid molecule, wherein the secondoligonucleotide comprises two regions which will hybridize underphysiological conditions to at least two of the two regions of the firstoligonucleotides which will not hybridize to the target nucleic acidmolecule, and wherein the multimeric nucleic acid inhibitory moleculecomprises both single-stranded and double-stranded regions.
 26. Themultimeric nucleic acid inhibitory molecule of claim 25, wherein thesequence that will not hybridize to the target nucleic acid molecule isseparated from the target binding nucleic acid segment by at least onenucleotide having a purine base, a pyrimidine base, or no base (abasic).27. The multimeric nucleic acid inhibitory molecule of claim 25, whereinthe target nucleic acid molecule is a long non-coding RNA or a shortnon-coding RNA.
 28. The multimeric nucleic acid inhibitory molecule ofclaim 25, wherein the first oligonucleotide is between from about 30nucleotides to about 200 nucleotides in length.
 29. The multimericnucleic acid inhibitory molecule of claim 25, wherein the secondoligonucleotide is between from about 6 nucleotides to about 50nucleotides in length.
 30. The multimeric nucleic acid inhibitorymolecule of claim 25, wherein the single-stranded region is capable ofbinding a target nucleic acid molecule under physiological conditions.31. The multimeric nucleic acid inhibitory molecule of claim 25, whereinthe single-stranded region is complementary to all or a portion of thetarget nucleic acid.
 32. The multimeric nucleic acid inhibitory moleculeof claim 27, wherein the short non-coding RNA is chosen from a microRNA,Piwi-interacting RNA, and small interfering RNA, and a ribosomal RNA.33. The multimeric nucleic acid inhibitory molecule of claim 25, whereinthe nucleic acid molecule comprises at least one modification chosenfrom, DNA, RNA, 2′OMe, 2′OAllyl, 2′O-propargyl, 2′O-alkyl, 2′ fluoro, 2′arabino, 2′ xylo, 2′ fluoro arabino, phosphorothioate,phosphorodithioate, 2′ amino, 5-alkyl-substituted pyrimidine,5-halo-substituted pyrimidine, alkyl-substituted purine,halo-substituted purine, bicyclic nucleotides, 2′MOE, LNA, andderivatives thereof.
 34. The multimeric nucleic acid inhibitory moleculeof claim 25, wherein the molecule further comprises one or morenucleotides on either end of the molecule.
 35. The multimeric nucleicacid inhibitory molecule of claim 25, wherein the target binding nucleicacid segment is about 60 to about 100% complementary to all or a portionof at least one target nucleic acid.
 36. A method for inhibiting thefunction of a target RNA or DNA molecule, the method comprisingcontacting the target RNA or DNA molecule with the nucleic acidinhibitory molecule of claim
 1. 37. A method for inhibiting the functionof a target RNA or DNA molecule, the method comprising contacting thetarget RNA or DNA molecule with the nucleic acid inhibitory molecule ofclaim
 25. 38. A method for producing a multimeric nucleic acidinhibitory molecule, comprising: obtaining at least a firstoligonucleotide and a second oligonucleotide, wherein the firstoligonucleotide comprises a target nucleic acid molecule binding region,and two or more regions which will not hybridize to the target nucleicacid molecule, wherein the second oligonucleotide comprises two regionswhich will hybridize under physiological conditions to at least two ofthe two or more regions of the first oligonucleotide which will nothybridize to the target nucleic acid molecule; admixing equal amounts ofthe first and second oligonucleotides in an appropriate buffer andallowing annealing of the two molecules, wherein the resultingmultimeric nucleic acid inhibitory molecule comprises bothsingle-stranded and double-stranded regions.
 39. A composite nucleicacid inhibitory molecule which comprises: a 5′ and/or 3′ non-nucleotideloop, and a target binding nucleic acid segment which is greater than 6nucleotides in length, wherein the target binding nucleic acid segmentis modified to increase binding affinity to its target.
 40. Thecomposite nucleic acid inhibitory molecule of claim 39, wherein thenon-nucleotide loop is chosen from polyethylene glycol, C2-C18 alkanediol, styrene, stilbene, triazole, tetrazole, nucleic acid, poly abasicnucleoside, polysaccharide, peptide, polyamide, hydrazone, oxyimine,polyester, disulfide, polyamine, polyether, peptide nucleic acid,cycloalkane, polyalkene, aryl, any combination thereof, and derivativesthereof.
 41. The composite nucleic acid inhibitory molecule of claim 40,wherein the polyethylene glycol is a polyethylene glycol derivative andwherein the polyethylene glycol derivative is hexa-ethylene glycol orpenta-ethylene glycol.
 42. The composite nucleic acid inhibitorymolecule of claim 39, wherein the loop is separated from the targetbinding nucleic acid segment by at least one nucleotide having a purinebase, a pyrimidine base, or no base (abasic).
 43. The composite nucleicacid inhibitory molecule of claim 39, wherein the target nucleic acidmolecule is a long non-coding RNA or a short non-coding RNA.
 44. Thecomposite nucleic acid inhibitory molecule of claim 42, wherein theshort non-coding RNA is chosen from a microRNA (miRNA), aPiwi-interacting RNA (piRNA), and a small interfering RNA (siRNA). 45.The composite nucleic acid inhibitory molecule of claim 39, wherein thenucleotides in the target binding nucleic acid segment comprises atleast one 2′ O-alkyl, LNA, 2′ fluoro, 2′ arabino, 2′ xylo, 2′ fluoroarabino, phosphorothioate, phosphorodithioate, 2′ amino, bicyclicnucleotide, 5-alkyl-substituted pyrimidine, 5-halo-substitutedpyrimidine, alkyl-substituted purine, or halo-substituted purine,halo-substituted purine, bicyclic nucleotides, 2′MOE, LNA, andderivatives thereof.
 46. The composite nucleic acid inhibitory moleculeof claim 39, wherein the target binding nucleic acid segment is about 60to about 100% complementary to all or a portion of at least one targetnucleic acid.
 47. A method for inhibiting the function of a target RNAor DNA molecule, the method comprising contacting the target RNA or DNAmolecule with the nucleic acid inhibitory molecule of claim
 39. 48. Amethod for treatment of a disease, comprising administering an amount ofthe inhibitory molecule of claims 1, 25, and 39 in an amount effectiveto treat a disease, wherein treatment of the disease comprises reducingthe symptoms of a disease.
 49. The method of claim 47, furthercomprising complexing the inhibitory molecule with a cellular deliveryagent.
 50. The method of claim 47, wherein the disease is chosen fromcancer, Alzheimer's disease, diabetes, and viral infections.
 51. Thecomposite nucleic acid inhibitory molecule of claim 1, furthercomprising a sterol moiety for self-delivery.
 52. The multimeric nucleicacid inhibitory molecule of claim 25, further comprising a sterol moietyfor self-delivery.